Recombinant P. falciparum merozoite protein-142 vaccine

ABSTRACT

In this application is the expression and purification of a recombinant  Plasmodium falciparum  (3D7) MSP-1 42 . The method of the present invention produces a highly purified protein which retains folding and disulfide bridging of the native molecule. The recombinant MSP-1 42  is useful as a diagnostic reagent, for use in antibody production, and as a vaccine.

[0001] This application claims the benefit of priority under 35 U.S.C.§119(e) from U.S. application serial No. 60/264,535 filed on Jan. 26,2001, still pending, and U.S. provisional application filed on Oct. 26,2001, still pending.

INTRODUCTION

[0002]Plasmodium falciparum is the leading cause of malaria morbidityand mortality. The World Health Organization estimates thatapproximately 200 million cases of malaria are reported yearly, with 3million deaths (World Health Organization, 1997, Wkly. Epidemiol. Rec.72:269-276). Although, in the past, efforts have been made to developeffective controls against the mosquito vector using aggressiveapplications of pesticides, these efforts ultimately led to thedevelopment of pesticide resistance. Similarly, efforts at treatment ofthe disease through anti-parasitic drugs led to parasitedrug-resistance. As the anti-vector and anti-parasite approaches failed,efforts became focused on malaria vaccine development as an effectiveand inexpensive alternative approach.

[0003] However, the complex parasitic life cycle has further confoundedthe efforts to develop efficacious vaccines for malaria. The parasite'slife cycle is divided between the mosquito-insect host and the humanhost. While in the human host, it passes through several developmentalstages in different organellar environments, i.e. the liver stage, thered blood stage. Although conceptually simple, in reality the problemsthat must be considered when designing subunit vaccines for malaria aregreat. Antigen diversity is a characteristic that must be taken intoaccount and includes a high degree of developmental stage specificity,antigenic variation and antigen polymorphism. Vaccine candidates havebeen identified from each of the parasite's developmental stages. Themajor merozoite surface protein-1, MSP-1, is among the leadingerythrocytic stage vaccine candidates (Diggs, et al, 1993, Parasitol.Today 9: 300-302). The objective of erythrocytic stage vaccines is todiminish the level of parasitemia in the bloodstream and thus reduce theseverity of disease.

[0004] Although the MSP-1 molecule has been studied extensively, itsfunction is not fully understood. There is evidence that MSP-1 binds toerythrocytes and may have a role in erythrocyte invasion (Perkins andRocco, 1988, J. Immunol. 141, 3190-3196; Holder, A. A., 1994,Parasitology 108 (Suppl.) S5-18).

[0005] MSP-1 is secreted as a membrane-anchored (Haldar et al., 1985, J.Biol. Chem. 260, 4969-4974) 195 kDa precursor that is proteolyticallyprocessed to products with nominal molecular masses of 83, 28-30, 38-45,and 42 kDa during merozoite development (Holder and Freem,an, 1984,Phils Trans R. Soc. Lond B. Bio. Sci. 307, 171-177; Lyon et al., 1987,J. Immunol, 138, 895-901; Holder et al., 1987, Parasitology 94,199-208). These protein fragments form a non-covalent complex on thesurface of merozoites (McBride and Heidrich, 1987, Parasitology 23,71-84; Lyon, et al., 1987, supra) that remain attached to the merozoitesurface through the C-terminal 42 kDa fragment (MSP-1₄₂). At the time oferythrocyte invasion MSP-1₄₂ is processed further to a 33 kDa fragmentand a 19 kDa C-terminal fragment (MSP-1₁₉) (Blackman, et al., 1991, Mol.Biochem. Parasitol. 49, 35-44) which is bound to the merozoite surfacethrough an N-glycosylphosphatidyl inositol anchor (GPI) (Haldar, et al.,1985, supra). This second proteolytic cleavage event results in theshedding of the non-covalent associated protein complex from themerozoite surface during invasion. During the invasion process, MSP-1₁₉is present on ring forms in the newly invaded erythrocyte (Blackman, etal., 1990, J. Exp. Med. 172, 379-382). The apparent structure of MSP-1₁₉is complex, containing 12 cysteines within a span of 100 amino acidresidues, and is arranged as two tandem domains that are homologous withepidermal growth factor (EGF) (Blackman, et al., 1991, supra; Morgan etal., 2000, J. Biomol. NMR 17, 337-347). Each putative EGF-domaincontains six cyteine residues that would form three disulfide bridgesper domain, which force the assembly of several well defineddiscontinous epitopes (Farley and Long, 1995, Exp. Parasitol. 80,328-332; McBride and Heidrich, 1987, supra; Uthaipibull et al, 2001, J.Mol. Biol. 307, 1381-1394).

[0006] Because age-dependent development of immunity to malaria is due,at least in part, to antibody against erythrocytic stage parasites(Cohen, S. et al., 1964, Nature 192, 733-737), a malaria vaccine shouldinduce effective antibodies against this developmental stage. Evidencesupporting the use of MSP-1₄₂ and MSP-1₁₉ in a malaria vaccine isextensive. MSP-1₁₉-specific mAbs inhibit P. falciparum growth in vitro(Blackman et al., 1990, supra) and passively protect mice againstinfection with P. yoelii (Majarian et al., 1984, J. Immunol. 132,3131-3137; Ling et al., 1994, Parasite Immunol. 16, 63-67). Immunizationof Aotus monkeys with native P. falciparum MSP-1 (Siddiqui, et al.,1987, Proc. Natl. Acad. Sci. USA 84, 3014-3018), or S. cerevisiaerecombinant MSP-1₁₉ (Kumar et al., 1995, Mol. Med. 1, 325-332; Egan etal., 2000, Infect. Immun. 68, 1418-1427; Stowers et al. 2001, TrendsParasitol. 17, 415-419), protect against a homologous challenge. E. coli-expressed P. yoelii MSP-1₉ (Burns et al., 1989, J. Immunol. 143,2670-2676) protects against a homologous challenge in rodent models.Antibodies raised against yeast MSP-1₁₉ grown in yeast weakly inhibitPlasmodium growth in vitro (Gozalo et al., 1998, Am. J. Trop. Med. Hyg.59, 991-997) however this antigen lacks correct structure and induces astrong allergic response (Keitel, W. A., 1999, Vaccine 18, 531-539).MSP-1₁₉ may not be an optimal vaccine because it does not induce strongT-helper cell responses (Quin et al., 2001, Eur. J. Immunol. 31, 72-81).Poor MSP-1₁₉ T-cell immunogenicity may be a consequence of itsstructural stability, which allows it to resist proteolysis, andtherefore to resist processing and presentation to the immune system.

[0007] Thus, MSP-1₄₂ may be a better choice as a vaccine candidate (Quinand Langhorne, 2001, Infect. Immun. 69, 2245-2251). Immunization ofAotus monkeys with baculovirus-expressed recombinant MSP-1₄₂, protectsagainst a homologous challenge and the anti-sera raised, inhibit P.falciparum growth in vitro, but do not result in sufficient yield andare not yet available in clinical grade ((Chang et al., 1996, Infect.Immun. 64, 253-261; Chang et al., 1992, J. Immunol. 149, 548-555).Monoclonal antibodies that either protect against infection in vivo(Burns et al., 1989, J. Immunol. 143, 2670-2676), or inhibit parasitegrowth in vitro (Blackman et al., 1990, supra), are specific fordiscontinuous epitopes since they do not react with disulfide-reducedMSP-1₁₉ (McBride and Heidrich, 1987, supra; Farley and Long, 1995, Exp.Parasitol. 80, 328-332). Thus, a recombinant vaccine produced from thispart of the MSP-1 will require correct disulfide-dependent conformationto elicit a protective antibody response.

[0008] Therefore, heterologous expression of recombinant molecules mustreplicate the conformation and structure of these proteins to induce anappropriate immune response. Heterologous expression of recombinant(MSP-1₄₂) from eukaryotic expression systems, i.e. baculovirus andyeast, have lead to recombinant molecules that possesspost-translational modifications due to N-glycosylation, and areexpressed poorly or are misfolded. Post-translational modification dueto N-glycosylation may be problematic for malaria vaccines becausemalaria parasites lack this ability.

[0009] Other attempts at producing MSP-1 in E. coli have not producedprotective vaccines (Kumar, S. et al., 1995, Molecular Medicine 1,325-332) due to problems with endotoxin contamination and possibly to aninability to establish correct disulfide bridging patterns.

[0010] Previous attempts show that not only is a good expression systemneeded for proper and sufficient expression of MSP-1₄₂ but, in addition,a good purification protocol is required which removes endotoxincontamination but which retains the proper folding of the antigen forpresentation to the immune system.

SUMMARY OF THE INVENTION

[0011] The present invention satisfies the needs discussed above. Thepresent invention provides a method for proper expression andpurification of the MSP-1₄₂ 3D7 allele. The method of the presentinvention results in elimination of contaminating proteins andconservation of the native folding and disulfide bridging of theprotein. Therefore, the essentially purified MSP-1₄₂ protein of thepresent invention retains proper conformation for optimal reactivity forvaccine and screening purposes.

[0012] Therefore, a major aim of the present invention resides in theproduction of large amounts of MSP-1₄₂ which maintain conformationalepitopes critical to epitope formation in pure form (>95% pure) fordiagnostic, prophylactic and therapeutic purposes.

[0013] This may not seem complicated but, as with most strategies forprotein purification, proved to be difficult and unpredictable. E. coliwas chosen as a host, even though it had gone out of favor, for tworeasons: (1) E. coli was known to produce high level of recombinantproteins and (2) recombinant proteins produced in E. coli are notglycosylated, which is consistent with the capabilities of malariaparasites. Several hurdles had to be overcome to achieve the desiredexpression level in soluble cytoplasmic form which can be sufficientlypurified from host cell proteins without sacrificing proper folding ofthe protein. Problems with E. coli endotoxin levels, antibioticresistance and the presence of non-MSP-1₄₂ contaminants had to beresolved.

[0014] The final expression construct, pETATPfMSP-1₄₂(3D7) (depositedwith ATCC under the Budapest Treaty on ______, accession number ______),was the product of a series of subclonings, with each successiveconstruction reducing the amount of expressed non-MSP-1 sequence. Theconstruction of a DNA vector expressing a P. falciparum 3D7 MSP-1₄₂molecule proceeded through several steps. A full-length fusion with E.coli thioredoxin at the N-terminus of MSP-1₄₂ was prepared by cloning inthe multiple cloning region of the pET32a expression vector (Construct#1, FIG. 1A, pET-Trx42). The expressed protein is identified in SEQ IDNO:1. Positive clones were transformed into the highly regulatable T7RNA polymerase expressing host. Mini-induction experiments wereconducted to optimize expression levels of several clones. In theseexperiments some variables that were investigated included inductiontemperature, concentration of inducer (IPTG), length of time ofinduction, and the influence of E. coli host background on levels ofexpression [BL21(DE3) versus AD494 (DE3)]. These variables have beenshown to affect the levels of expression and the partitioning of proteinin either soluble or insoluble fractions. SDS-PAGE and immunoblottinganalysis of crude extracts from cells induced at 37° C. showed that thefull length fusion, trxA-MSP-1₄₂ (Construct #1, FIG. 1A) comprisedgreater than 20% of the total E. coli protein. However, following celllysis, all of the fusion protein partitioned into the insoluble fractionand was associated with inclusion bodies. This situation is often thecase with heterologous proteins that are expressed at high levels in E.coli.

[0015] Lowering the culture temperature sequentially from 37° C. to 25°C. during induction of expression resulted in increasing levels ofsoluble fusion protein in the post-sonication supernatant. By increasingthe soluble protein at this stage, a urea solubilization and refoldingstep is avoided thereby assuring more native folding of the protein. Thepost sonication soluble supernatant was applied to a Ni⁺²=NTA agaroseaffinity column (QIAGEN) and bound protein was eluted with stepwiseincreasing gradients of imidazole. The expressed thioredoxin-MSP-1₄₂fusion products from these cells was reactive with mAb 5.2 (see Table 1)on immunoblots. Our data suggested that this expression system wouldprovide sufficient levels of recombinant protein for development as adiagnostic and vaccine antigen, providing proper covalent disulfidebridging could be achieved.

[0016] A second construct was designed (Construct #2, pET(50)MSP-1₄₂) todelete the E. coli trxA gene (thioredoxin protein) from Construct #1(thioredoxin-MSP-1₄₂ fusion). This product was developed as analternative to the full-length thioredoxin fusion to address potentialFDA regulatory concerns with a thioredoxin-MSP-1₄₂ fusion proteinvaccine. The product formed retains the His6-tag for affinitypurification and an additional vector encoded sequence (approximately 50amino acids) which include an enterokinase cleavage site, and S-peptidetag, and the thrombin cleavage site fused to the N-terminus of MSP-1₄₂.The expressed product is identified in SEQ ID NO:2. The levels ofexpression from this construct were estimated at approximately 5-10% ofthe total E. coli protein from crude cell lysates and protein waspurified to near homogeneity (>85%) with two consecutive passes over aNi⁺²-NTA agarose resin. TABLE 1 MSP-119-Specific Monoclonal AntibodiesImmunizing Antibody parasite Stages Epitope/location Ref.  2.2-7 Thai-K1smr* Conserved/EGF-like McKay domain 1 12.8-2-1 Thai-T9-96 smrConserved/EGF-like Conway (K1 like) domain 1  7.5-1 Thai-K1 smrSemi-conserved/ McBride EGF-like domain 1 12.10-5-1 Thai-T9-96 smrSemi-conserved, Blackman (K1 like) EGF-like domain 1 & 2  5.2Uganda-Palo Alto smr Semi-conserved, Chang (3D7 like) EGF-like domain 17F1 Malayan Camp sm MSP-133Strain Lyon (3D7 like) Specific Polyclonal3D7 MSP-1-42 smr Many epitopes Angov

[0017] Polyclonal rabbit MSP-142 antibody was prepared at Walter ReedArmy Institute of Research Department of Immunology, by immunization ofrabbits with recombinant E. coli-expressed MSP-142 (3D7) adjuvanted withComplete Freunds Adjuvant.

[0018] Since the levels of expression and apparent protein folding ofConstruct #2 suggested that a correctly folded non-thioredoxin-fusedMSP-1₄₂ was expressible, a third construct was developed to remove theentire vector non-MSP-1 encoded sequence fused at the N-terminus ofConstruct #2. This upstream gene sequence was deleted and was replacedwith an annealed oligonucleotide linker to regenerate the His6-tag(Construct #3, pET42A). Therefore, Construct #3 contains 9 non-MSP-1₄₂amino acids that include the His6 and 3 linker amino acids. Theexpressed product is identified in SEQ ID NO:3. The non-fused MSP-1₄₂molecule from this construct is produced to adequate levels (2-5%) ofthe total E. coli protein and is correctly folded based onimmunoreactivity with a series of MSP-1₁₉ specific mAbs (See Table 1).Potential regulatory concerns over selection in the presence ofampicillin resulted in a final modification on the His6-MSP-1₄₂construct (Construct #3) that included the gene for tetracycline(Construct #4, pET-IEGR-MSP142(AT), FIG. 1B). Therefore, the plasmiddesignated as Construct #4 (also His6-MSP-1₄₂, but selectable withtetracycline) can be selected in the presence of tetracycline aloneduring large-scale fermentation or with ampicillin, as necessary. Theexpressed protein of Construct #4 is identified in SEQ ID NO:3. Thefinal plasmid pET(AT)PfMSP-1₄₂(3D7) was created by removing the residualFactor Xa cleavage site. Constructs 1-4 can be used to produce fusionproteins with MSP-1₄₂ as a source of soluble antigen.

[0019] Intensive investigation of variables that affect the efficiencyof fermentation and induction of expression were required to optimizeHis6-MSP-1₄₂ expression. Some variables which have a significant effecton target protein yields and bear upon purification strategies includethe effects of media composition, amount of inducer necessary,temperature at which inducer is added to the cultures, and length oftime of induction, to name a few.

[0020] A low temperature of induction was necessary in order to obtainsoluble protein. The temperature of the cultures had to be reduced from37° C. to about 25° C. prior to induction. At all higher temperatures,protein was found in inclusion bodies and difficult to isolate.Similarly, we found that the time of induction was important for properand maximal expression of the protein. The length of IPTG induction wasmost advantageous at 2-3 hours. Induction for less time resulted insuboptimal protein synthesis and induction for more time resulted inloss of product due to lysis and protein degradation.

[0021] Cells were suspended in lysis buffer and lysed bymicrofluidization (Microfluidics) in one pass while the temperature ofthe sample was maintained below 10° C. at all times to reduceproteolysis. The lysate was centrifuged and the pellets and supernateswere evaluated by immunoblotting.

[0022] The clarified lysate was then purified by several methods. Thefirst method is using the ability of the His6-tag sequence expressed asa short N-terminal fusion on the target protein to bind to divalentcations, i.e. nickel, immobilized onto matrices. The w/v cell paste toresin ratio were varied to optimize yield and purity of the product andto minimize cost.

[0023] After elution of the protein from the Ni²⁺ chelate resin, it isallowed to incubate at 0-4° C. overnight to promote disulfide bridgeformation. This step is required because protein disulfie bridgeformation does not occur readily in the cytoplasm of the E. coli BL21DE3 expression host due to the reducing nature of this environment. Thisexplains our observation that monoclonal antibodies known to react withproperly folded disulfide bridged MSP-1₄₂ do not react with this proteineither in cell lysates, or with partially purified recombinant MSP-1₄₂immediately after its elution from the Ni⁺² NTA affinity column.Incubating the eluted protein at 0-4° C. for up to 48 hours followingthe Ni⁺² chromatography promotes proper disulfide bridge fromationbecause after this incubation, all of the monoclonal antibodies nowreact with the antigen. These data suggest that at the time of lysis andchromatography on the Ni⁺² NTA affinity column the protein is probablyfolded properly through ionic and hydrophobic interactions, but that thedisulfide bridges do not form until the protein is maintained in anoxidative environment over the observed period of time.

[0024] The eluted sample was then applied to a Q ion exchangechromatography column. Binding of the sample to the column was checkedat varying pH values ranging from pH 6.2 to 9.9. In order to define a pHcondition at which the protein would bind to the Q resin, we variedbuffer types, e.g. phosphate (pH 6-9) vs. glycine (pH 8.5-11) vs.citrate (pH 4-5-6). Under the final conditions selected, MSP-1₄₂partitioned into the flow through and most residual host cell remainedproteins bound to the column.

[0025] Finally, CM ion exchange chromatography was used to remove lowlevels of residual E. coli protein and endotoxin. Variables optimizedincluded the binding of protein to the column, the salt concentration ofthe washing and eluting solution.

[0026] The purified P. falciparum MSP-1₄₂ was used as a vaccine alongwith an adjuvant, for example, ADJUVANT B, and was found to elicitmalaria specific antibody responses in monkeys. More importantly,vaccination with the MSP-1₄₂ elicited neutralizing antibodies andprotects monkeys against a malaria infection.

[0027] Therefore, it is an object of the present invention to provide arecombinant P. falciparum MSP-1₄₂ for use in diagnostic assays and forproduction of antibodies.

[0028] It is another object of the present invention to providecompositions comprising purified recombinant P. falciparum MSP-1₄₂.

[0029] It is yet another object of the present invention to providenovel vector constructs for recombinantly expressing P. falciparumMSP-1₄₂, as well as host cells transformed with said vector.

[0030] It is also an object of the present invention to provide a methodfor producing and purifying recombinant P. falciparum MSP-1₄₂ proteincomprising:

[0031] growing a host cell containing a vector expressing P. falciparumMSP-1₄₂ proteins in a suitable culture medium,

[0032] causing expression of said vector sequence as defined above undersuitable conditions for production of soluble protein and,

[0033] lysing said transformed host cells and recovering said MSP-1₄₂protein such that it retains its native folding and is essentially freeof host toxins.

[0034] It is also an object of the present invention to providediagnostic and immunogenic uses of the recombinant P. falciparum MSP-1₄₂protein of the present invention, as well as to provide kits fordiagnostic use for example in malaria screening and confirmatoryantibody tests.

[0035] It is also an object of the present invention to providemonoclonal or polyclonal antibodies, more particularly human monoclonalantibodies or mouse monoclonal antibodies which are humanized, whichreact specifically with MSP-1₄₂ epitopes, either comprised in peptidesor conformational epitopes comprised in recombinant proteins.

[0036] It is also an object of the present invention to provide possibleuses of anti-MSP-1₄₂ monoclonal antibodies for malaria antigen detectionor for therapy of chronic malaria infection.

[0037] It is yet another object of the present invention to provide amalaria vaccine comprising MSP-1₄₂.of the present invention, in anamount effective to elicit an immune response in an animal against P.falciparum; and a pharmaceutically acceptable diluent, carrier, orexcipient.

[0038] It is another object of the present invention to provide a methodfor eliciting in a subject an immune response against malaria, themethod comprising administering to a subject a composition comprisingMSP-1₄₂ of the present invention. In one aspect of the invention, theDNA vaccine is delivered along with an adjuvant, for example ADJUVANT B.

[0039] It is another object of the present invention to provide a methodfor preventing malaria infection in an animal comprising administeringto the animal the MSP-1₄₂ of the present invention.

[0040] The vaccine according to the present invention is inherentlysafe, is not painful to administer, and should not result in adverseside effects to the vaccinated individual.

[0041] The present invention also provides vectors for the production ofa recombinant MSP-1₄₂, host cells containing the vectors, a method forfermenting and inducing the host cells, and a method for isolating andpurifying the recombinant protein. Also provided is a method for bulkfermentation and expression of MSP-1₄₂.

[0042] All the objects of the present invention are considered to havebeen met by the embodiments as set out below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043]FIG. 1A: pET32a plasmid map.

[0044]FIG. 1B: pET(AT)PfMSP-1₄₂ plasmid map.

[0045]FIG. 2A, 2B and 2C: SDS-PAGE analysis of MSP-1₄₂ duringPurification and of the Final Product (FMP1). Protein detected byCoomassie Blue staining (2A and 2B) and immunoblotting (2C). 2A,non-reduced samples: lane 1, nickel chelate eluate; lane 2, Q flowthrough; lane 3 CM eluate. 2B, 10 ug FMP1 electrophoresed undernon-reduced (lane 1) or reduced (lane 2) conditions. 2C, immunoblottingof 1 ug of FMP electrophoresed under non-reducing: lane 1 mAb 7F1; lane2, mAb 12.10; lane 3, rabbit anti-E. coli antiserum.

[0046]FIG. 3: FMP1 Stability. Commassie Blue stained non-reducedSDS-PAGE gel with 10 ug samples of FMP1 stored for 18 months undervarious conditions. Lane 1, −80° C.; lane 2, −20° C.; lane 3, 4° C.

[0047]FIG. 4: FMP1 Immunogenicity. Rhesus monkeys were boosted 1, 3, 5,and 7 months after priming with FMP1/ADJUVANT B (rectangles) orFMP1/alum (circles). Sera were collected just prior to immunization(arrows) and two weeks after each immunization. In the case of theADJUVANT B cohort serum collection continued monthly for 9 months.Antibody titers were measured by IFA (filled symbols) and kinetic ELISA(open symbols).

[0048]FIG. 5: Merozoite Invasion Inhibition. Triplicate synchronouscultures of P. falciparum schizont-infected erythrocytes were incubatedfor 24 hours with the IgG fractions of pre-immune (open diamonds) orimmune (filled diamonds) IgG fractions from rabbits immunized with FMP1in Fruends adjuvents. IgG was tested at doses of 200, 400, and 800 ug/mland parasite growth was quantified by counting 5000 erythroctyes or 100parasitized erythrocytes, whichever occurred first. Data are shown asthe mean parasitemia and 95% confidence interval.

[0049]FIG. 6: CMI responses to FMP1/ADJUVANT B. Peripheral bloodmononuclear cells (PBMC) were stimulated with FMP1 (top) or P.falciparum parasitized erythrocytes (PBRC, bottom) and proliferation wasmeasured by 3H-thymidine uptake. Each panel contains data from allindividuals within a vaccine group and individuals are differentiated bysymbol. Samples were collected prior to immunization (pre) and two weeksfollowing each immunization (post 1, post 2, post 3).

DETAILED DESCRIPTION

[0050] In the description that follows, a number of terms used inrecombinant DNA, parasitology and immunology are extensively utilized.In order to provide a clearer and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided.

[0051] In general, an ‘epitope’ is defined as a linear array of 3-10amino acids aligned along the surface of a protein. In a linear epitope,the amino acids are joined sequentially and follow the primary structureof the protein. In a conformational epitope, residues are not joinedsequentially, but lie linearly along the surface due to the conformation(folding) of the protein. With respect to conformational epitopes, thelength of the epitope-defining sequence can be subject to widevariations. The portions of the primer structure of the antigen betweenthe residues defining the epitope may not be critical to the structureof the conformational epitope. For example, deletion or substitution ofthese intervening sequences may not affect the conformational epitopeprovided sequences critical to epitope conformation are maintained (e.g.cysteines involved in disulfide bonding, glycosylation sites, etc.). Aconformational epitope may also be formed by 2 or more essential regionsof subunits of a homo-oligomer or hetero-oligomer. As used herein,‘epitope’ or ‘antigenic determinant’ means an amino acid sequence thatis immunoreactive. As used herein, an epitope of a designatedpolypeptide denotes epitopes with the same amino acid sequence as theepitope in the designated polypeptide, and immunologic equivalentsthereof. Such equivalents also include strain, subtype (=genotype), ortype(group)-specific variants, e.g. of the currently known sequences orstrains belonging to Plasmodium such as 3D7, FVO and CAMP, or any otherknown or newly defined Plasmodium strain.

[0052] The term ‘solid phase’ intends a solid body to which theindividual P. falciparum antigen is bound covalently or by noncovalentmeans such as hydrophobic, ionic, or van der Waals association.

[0053] The term ‘biological sample’ intends a fluid or tissue of amammalian individual (e.g. an anthropoid, a human), reptilian, avian, orany other zoo or farm animal that commonly contains antibodies producedby the individual, more particularly antibodies against malaria. Thefluid or tissue may also contain P. falciparum antigen. Such componentsare known in the art and include, without limitation, blood, plasma,serum, urine, spinal fluid, lymph fluid, secretions of the respiratory,intestinal or genitourinary tracts, tears, saliva, milk, white bloodcells and myelomas. Body components include biological liquids. The term‘biological fluid’ refers to a fluid obtained from an organism. Somebiological fluids are used as a source of other products, such asclotting factors (e.g. Factor VlIl;C), serum albumin, growth hormone andthe like.

[0054] The term ‘immunologically reactive’ means that the antigen inquestion will react specifically with anti-MSP-1 antibodies present in abody component from a malaria infected individual.

[0055] The term ‘immune complex’ intends the combination formed when anantibody binds to an epitope on an antigen.

[0056] The term ‘MSP-1₄₂’ as used herein refers to the polymorphicC-terminal 42 kDa protein fragment or polypeptide resulting from theprocessing by proteases of the 195 kDa membrane-anchored MSP-1precursor. During merozoite invasion, the 42 kDa fragment is subjectedto secondary processing producing a 33-kDa fragment (MSP-1₃₃) and a 19kDa C-terminal fragment, (MSP-1₁₉) which remains attached via GPI to thesurface of the invading merozoite. The MSP-1₄₂ protein extends fromapproximately amino acid (aa) 1327 to about aa 1700 of the full-lengthprecursor protein (Genbank accession #z35327).

[0057] The term ‘MSP-1₄₂’ as used herein also includes analogs andtruncated forms that are immunologically cross-reactive with naturalMSP-1₄₂. By ‘MSP-1₄₂’ is intented MSP-1₄₂ from other strains ofPlasmodium, or any other newly identified strain of Plasmodium.

[0058] The term ‘homo-oligomer’ as used herein refers to a complex ofMSP-1₄₂ containing more than one MSP-1₄₂ monomer, e.g. MSP-1₄₂/MSP-1₄₂dimers, trimers or tetramers, or any higher-order homo-oligomers ofMSP-1₄₂ are all ‘homo-oligomers’ within the scope of this definition.The oligomers may contain one, two, or several different monomers ofMSP-1₄₂ obtained from different strains of Plasmodium falciparumincluding for example 3D7, Camp, FVO, and others. Such mixed oligomersare still homo-oligomers within the scope of this invention, and mayallow more universal diagnosis, prophylaxis or treatment of malaria.

[0059] The term ‘purified’ as applied to proteins herein refers to acomposition wherein the desired protein comprises at least 35% of thetotal protein component in the composition. The desired proteinpreferably comprises at least 40%, more preferably at least about 50%,more preferably at least about 60%, still more preferably at least about70%, even more preferably at least about 80%, even more preferably atleast about 90%, and most preferably at least about 95% of the totalprotein component. The composition may contain other compounds such ascarbohydrates, salts, lipids, solvents, and the like, without affectingthe determination of the percentage purity as used herein. An ‘isolated’MSP-1₄₂ protein intends a Plasmodium protein composition that is atleast 35% pure.

[0060] The term ‘essentially purified proteins’ refers to proteinspurified such that they can be used for in vitro diagnostic methods andas a prophylactic compound. These proteins are substantially free fromcellular proteins, vector-derived proteins or other Plasmodiumcomponents. The proteins of the present invention are purified tohomogeneity, at least 80% pure, preferably, 90%, more preferably 95%,more preferably 97%, more preferably 98%, more preferably 99%, even morepreferably 99.5%.

[0061] The term ‘recombinantly expressed’ used within the context of thepresent invention refers to the fact that the proteins of the presentinvention are produced by recombinant expression methods be it inprokaryotes, or lower or higher eukaryotes as discussed in detail below.

[0062] The term ‘lower eukaryote’ refers to host cells such as yeast,fungi and the like. Lower eukaryotes are generally (but not necessarily)unicellular. Preferred lower eukaryotes are yeasts, particularly specieswithin Saccharomyces. Schizosaccharomyces, Kluveromyces, Pichia (e.g.Pichia pastoris), Hansenula (e.g. Hansenula polymorpha, Yarowia,Schwaniomyces, Schizosaccharomyces, zygosaccharomyces and the like.Saccharomyces cerevisiae, S. carlsberoensis and K. lactis are the mostcommonly used yeast hosts, and are convenient fungal hosts.

[0063] The term ‘prokaryotes’ refers to hosts such as E. coli ,Lactobacillus, Lactococcus, Salmonella, Streptococcus, Bacillus subtilisor Streptomyces. Also these hosts are contemplated within the presentinvention.

[0064] The term ‘higher eukaryote’ refers to host cells derived fromhigher animals, such as mammals, reptiles, insects, and the like.Presently preferred higher eukaryote host cells are derived from Chinesehamster (e.g. CHO), monkey (e.g. COS and Vero cells), baby hamsterkidney (BHK), pig kidney (PK15), rabbit kidney 13 cells (RK13), thehuman osteosarcoma cell line 143 B, the human cell line HeLa and humanhepatoma cell lines like Hep G2, and insect cell lines (e.g. Spodopterafrugiperda). The host cells may be provided in suspension or flaskcultures, tissue cultures, organ cultures and the like. Alternativelythe host cells may also be transgenic animals.

[0065] The term ‘polypeptide’ refers to a polymer of amino acids anddoes not refer to a specific length of the product; thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide. This term also does not refer to or exclude post-expressionmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like. Included within thedefinition are, for example, polypeptides containing one or moreanalogues of an amino acid (including, for example, unnatural aminoacids, PNA, etc.), polypeptides with substituted linkages, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring.

[0066] The term ‘recombinant polynucleotide or nucleic acid’ intends apolynucleotide or nucleic acid of genomic, cDNA, semisynthetic, orsynthetic origin which, by virtue of its origin or manipulation: (1) isnot associated with all or a portion of a polynucleotide with which itis associated in nature, (2) is linked to a polynucleotide other thanthat to which it is linked in nature, or (3) does not occur in nature.

[0067] The term ‘recombinant host cells’, ‘host cells’, ‘cells’, ‘celllines’, ‘cell cultures’, and other such terms denoting microorganisms orhigher eukaryotic cell lines cultured as unicellular entities refer tocells which can be or have been, used as recipients for a recombinantvector or other transfer polynucleotide, and include the progeny of theoriginal cell which has been transfected. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.

[0068] The term ‘replicon’ is any genetic element, e.g., a plasmid, achromosome, a virus, a cosmid, etc., that behaves as an autonomous unitof polynucleotide replication within a cell; i.e., capable ofreplication under its own control.

[0069] The term ‘vector’ is a replicon further comprising sequencesproviding replication and/or expression of a desired open reading frame.

[0070] The term ‘control sequence’ refers to polynucleotide sequenceswhich are necessary to effect the expression of coding sequences towhich they are ligated. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and terminators; ineukaryotes, generally, such control sequences include promoters,terminators and, in some instances, enhancers. The term ‘controlsequences’ is intended to include, at a minimum, all components whosepresence is necessary for expression, and may also include additionalcomponents whose presence is advantageous, for example, leader sequenceswhich govern secretion.

[0071] The term ‘promoter’ is a nucleotide sequence which is comprisedof consensus sequences which allow the binding of RNA polymerase to theDNA template in a manner such that mRNA production initiates at thenormal transcription initiation site for the adjacent structural gene.

[0072] The expression ‘operably linked’ refers to a juxtapositionwherein the components so described are in a relationship permittingthem to function in their intended manner. A control sequence ‘operablylinked’ to a coding sequence is ligated in such a way that expression ofthe coding sequence is achieved under conditions compatible with thecontrol sequences.

[0073] An ‘open reading frame’ (ORF) is a region of a polynucleotidesequence which encodes a polypeptide and does not contain stop codons;this region may represent a portion of a coding sequence or a totalcoding sequence.

[0074] A ‘coding sequence’ is a polynucleotide sequence which istranscribed into mRNA and/or translated into a polypeptide when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a translation start codon at the5′-terminus and a translation stop codon at the 3′-terminus. A codingsequence can include but is not limited to mRNA, DNA (including cDNA),and recombinant polynucleotide sequences.

[0075] The term ‘immunogenic’ refers to the ability of a substance tocause a humoral and/or cellular response, whether alone or when linkedto a carrier, in the presence or absence of an adjuvant.‘Neutralization’ refers to an immune response that blocks theinfectivity, either partially or fully, of an infectious agent. A‘vaccine’ is an immunogenic composition capable of eliciting protectionagainst malaria, whether partial or complete. A vaccine may also beuseful for treatment of an infected individual, in which case it iscalled a therapeutic vaccine.

[0076] The term ‘therapeutic’ refers to a composition capable oftreating malaria infection. The term ‘effective amount’ for atherapeutic or prophylactic treatment refers to an amount ofepitope-bearing polypeptide sufficient to induce an immunogenic responsein the individual to which it is administered, or to otherwisedetectably immunoreact in its intended system (e.g., immunoassay).Preferably, the effective amount is sufficient to effect treatment, asdefined above. The exact amount necessary will vary according to theapplication. For vaccine applications or for the generation ofpolyclonal antiserum/antibodies, for example, the effective amount mayvary depending on the species, age, and general condition of theindividual, the severity of the condition being treated, the particularpolypeptide selected and its mode of administration, etc. It is alsobelieved that effective amounts will be found within a relatively large,non-critical range. An appropriate effective amount can be readilydetermined using only routine experimentation. Preferred ranges ofMSP-1₄₂ for prophylaxis of malaria disease are about 0.01 to 1000ug/dose, more preferably about 0.1 to 100 ug/dose, most preferably about10-50 ug/dose. Several doses may be needed per individual in order toachieve a sufficient immune response and subsequent protection againstmalaria.

[0077] More particularly, the present invention contemplates essentiallypurified MSP-1₄₂ and a method for isolating or purifying recombinantMSP-1₄₂ protein, characterized in that upon lysing the transformed hostcells to isolate the recombinantly expressed protein, the disulfidebonds necessary for proper folding of the protein are preserved.

[0078] The term ‘MSP-1₄₂’ refers to a polypeptide or an analogue thereof(e.g. mimotopes) comprising an amino acid sequence (and/or amino acidanalogues) defining at least one MSP-1₄₂ epitope. Typically, thesequences defining the epitope correspond to the amino acid sequence ofMSP-1₄₂ region of P. falciparum (either identically or via substitutionof analogues of the native amino acid residue that do not destroy theepitope). The MSP-1₄₂ protein corresponds to a nucleotide sequenceidentified in SEQ ID NO:5 and spans from amino acid 1327 to 1701 ofMSP-1 3D7 allele (SEQ ID NO:6). Upon expression in the parasite system(non-glycosylated), it is believed to have an approximate molecularweight of 42 kDa as determined by SDS-PAGE. It is understood that theseprotein endpoints are approximations (e.g. the carboxy terminal end ofMSP-1₄₂ could lie somewhere in the 1700 to 1720 amino acid region). Theabsolute C-terminus is not defined due to the post-translationalmodification that transfers MSP-1 to a GPI lipid membrane anchor.

[0079] The MSP-1₄₂ antigen used in the present invention is preferably afull-length protein, or a substantially full-length version, i.e.containing functional fragments thereof (e.g. fragments which are notmissing sequence essential to the formation or retention of an epitope).Furthermore, the P. falciparum antigen of the present invention can alsoinclude other sequences that do not block or prevent the formation ofthe conformational epitope of interest. The presence or absence of aconformational epitope can be readily determined though screening theantigen of interest with an antibody as described in the Examples below(polyclonal serum or monoclonal to the conformational epitope) andcomparing its reactivity to that of a denatured version of the antigenwhich retains only linear epitopes (if any).

[0080] The P. falciparum antigen of the present invention can be made byany recombinant method that provides the epitope of interest. Forexample, recombinant expression in E. coli is a preferred method toprovide non-glycosylated antigens in ‘native’ conformation. This is mostdesirable because natural P. falciparum antigens are not glycosylated.Proteins secreted from mammalian cells may contain modificationsincluding galactose or sialic acids which may be undesirable for certaindiagnostic or vaccine applications. However, it may also be possible andsufficient for certain applications, as it is known for proteins, toexpress the antigen in other recombinant hosts such as baculovirus andyeast or higher eukaryotes, as long as glycosylation is inhibited.

[0081] The proteins according to the present invention may be secretedor expressed within compartments of the cell. Preferably, however, theproteins of the present invention are expressed within the cell and arereleased upon lysing the cells.

[0082] It is also understood that the isolates used in the examplessection of the present invention were not intended to limit the scope ofthe invention and that an equivalent sequence from a P. falciparumisolate from another allele, e.g. FVO, or CAMP, can be used to produce arecombinant MSP-1₄₂ protein using the methods described in the presentapplication. Other new strains of Plasmodium may be a suitable source ofMSP-1₄₂ sequence for the practice of the present invention.

[0083] The MSP-1₄₂ protein of the present invention is expressed as partof a recombinant vector. The present invention relates more particularlyto the MSP-1₄₂ nucleic acid sequence in recombinant nucleic acidspETATpfMSP-1₄₂(3D7) as represented in SEQ ID NO:7 or parts thereof. TheMSP-1₄₂ genomic sequence was cloned into pET32a from Novagen (Madison,Wis.). This plasmid comprises, in sequence, a T7 promoter, optionally alac operator, a ribosome binding site, restriction sites to allowinsertion of the structural gene and a T7 terminator sequence. Othervectors provided include pET-Trx42, pET(50)MSP-1₄₂, pET42A,pET-IEGR-MSP-1₄₂ (AT) all described below in Materials and Methods.Examples of other plasmids which contain the T7 inducible promoterinclude the expression plasmids pET-17b, pET-11a, pET-24a-d(+), andpEt-9a, all from Novagen (Madison, Wis.); see the Novagen catalogue.

[0084] The present invention also contemplates host cells transformedwith a recombinant vector as defined above. In a preferred embodiment,E. coli strain BL21 (DE3) (F-ompT hsdSB(rB-mB-) gal dcm (DE3)) isemployed. The above plasmids may be transformed into this strain orother strains of E. coli having the following characteristics: a T7 RNApolymerase rec gene, Lon, ompT protease mutants or any other E. coliwith a protease deficiency such as E. coli origami. Preferably, the hostincludes BL21(DE3) and any of its precursors. Other host cells such asinsect cells can be used taking into account that other cells may resultin lower levels of expression.

[0085] Eukaryotic hosts include lower and higher eukaryotic hosts asdescribed in the definitions section. Lower eukaryotic hosts includeyeast cells well known in the art. Higher eukaryotic hosts mainlyinclude mammalian cell lines known in the art and include manyimmortalized cell lines available from the ATCC, inluding HeLa cells,Chinese hamster ovary (CHO) cells, Baby hamster kidney (BHK) cells,PK15, RK13 and a number of other cell lines. MSP-1₄₂ expressed in thesecells will be glycosylated unless the cells have been altered such thatglycosylation of the recombinant protein is not possible. It is expectedthat when producing MSP-1₄₂ in a eukaryotic expression system, extensiveinvestigation into methods for expressing, isolating, purifying, andcharacterizing the protein would be required as eukaryotic cellspost-translationally modify this protein and this would alter proteinstructure and immunogenicity.

[0086] Methods for introducing vectors into cells are known in the art.Please see e.g., Maniatis, Fitsch and Sambrook, Molecular Cloning; ALaboratory Manual (1982) or DNA Cloning, Volumes I and II (D. N. Glovered. 1985) for general cloning methods. Host cells provided by thisinvention include E. coli containing pET-Trx42, E. coli containingpET(50)MSP-1₄₂, and E. coli containing pET42A, E. coli containingPET-IEGR-MSP-1₄₂(AT), and E. coli containing pET(AT)PfMSP-1₄₂(3D7).

[0087] A preferred method for isolating or purifying MSP-1₄₂ as definedabove is further characterized as comprising at least the followingsteps:

[0088] (i) growing a host cell as defined above transformed with arecombinant vector expressing MSP-1₄₂ proteins in a suitable culturemedium,

[0089] (ii) causing expression of said vector sequence as defined aboveunder suitable conditions for production of a soluble protein,

[0090] (iii) lysing said transformed host cells and recovering saidMSP-1₄₂ protein such that it retains its native conformation and isessentially pure.

[0091] Once the host has been transformed with the vector, thetransformed cells are grown in culture in the presence of the desiredantibiotic. For FDA regualtory purposes, it is preferable to usetetracycline or kanamycin. When cells reach optimal biomass density, inthis case about 0.4 OD 600 in small culture flasks or 4-6 OD 600 in bulkfermentors, the cells are induced to produce the recombinant protein.The inventors have found after trial and error that for expression of asoluble MSP-1₄₂, it was necessary to cool the culture to a range ofabout 10° C.-20° C., more preferably about 15° C.-28° C., mostpreferably about 24 to 26° C. prior to induction. The concentration ofinducer, i.e. IPTG, added affects the maximal protein synthesis. It wasfound that a concentration of 0.1 mM IPTG was best, however, a range of0.05 to 0.5 mM would be sufficient to produce 80-100% of maximal.

[0092] The cells were then collected and lysed to release therecombinant protein. Preferably, lysis should occur at a paste to bufferratio of 1:3 w/v to reduce viscosity and volume of sample loaded onNi-NTA column. Preferably, lysis is in the presence of imidazole whichreduces non specific binding of E. coli protein to Ni resin, andbenzonase which is able to digest E. coli nucleic acids at a reducedtemperature. Lysis is preferably at a temperature of about 0° C.-24° C.,more preferably about 5-15° C. in order to retain native folding of theMSP-1₄₂ protein and to reduce proteolysis. A high salt concentration ofabout 0.5-1.0 M is preferable. Salts used include NaCl or othermonovalent ions.

[0093] Preferably, the E. coli endotoxin is separated and removed fromthe recombinant protein. This can be done several ways. For MSP-1₄₂,endotoxin was removed by applying to a Ni⁺²-NTA column. The removal ofendotoxin depended on washing at low pH, about 5.8 to 6.5, preferablyabout pH 6.2, in high salt, about 0.5 to about 1.0 mM, preferably about500 mM NaCl at a flow rate of about 20 to about 35 ml/min, preferablyabout 30 ml/min. The cell paste to resin ratio can be about 5:1 to about7:1 w/v, preferably about 6:1 w/v. The recombinant protein can be elutedby addition of high pH buffer of about 7.5 to about 8.5, preferablyabout pH 8.0, in a phosphate buffer of about 10-20 mM, more preferablyabout 10 mM sodium phosphate buffer.

[0094] At this point the recombinant protein is about 50% pure. Iffurther purity is required, ion-exchange chromatography can be utilized.The column is preferably with an ionic character such that a pH toreduce protein binding and promote endotoxin and nucleic acid bindingcan be used.

[0095] Finally, the flow through sample (about 0.1 mg/ml), can besubjected to further ion exchange chromatography for furtherconcentration and purification. The MSP-1₄₂ of the present invention wassubjected to CM ion exchange chromatography. The pH of the buffer can beabout 5.2 to about 6.2, preferably about 6.0. The salt concentration isabout 25 mM to about 50 mM, preferably about 35 mM.

[0096] The bulk process for the isolation of purified MSP-1₄₂ differslittle from the process described above. The concentration of imidazoleis changed to about 120 mM to about 200 mM, preferably about 160 mM inorder to specifically elute MSP-1₄₂.

[0097] The present invention further relates to a composition comprisingat least one of the following

[0098] MSP-1₄₂ peptides as listed in Table 3:

[0099] MSP-1₄₂ alone (SEQ ID NO:5) spanning amino acids to 1326-1701 ofMSP-1,

[0100] MSP-1₄₂ with thioredoxin from vector pET-Trx 42 (SEQ ID NO:1);

[0101] MSP-1₄₂ without thioredoxin from vector pET(5)MSP-1₄₂ (SEQ IDNO:2);

[0102] MSP-1₄₂ plus His6tag produced from vector pET42A (SEQ ID NO:3)

[0103] MSP-1₄₂ without Factor Xa from vector PET-IEGR-MSP-1₄₂ (AT) (SEQID NO:3);

[0104] MSP-1₄₂ plus 17 amino acids at N-terminal in final construct (SEQID NO:3), from construct pET(AT)PfMSP-1₄₂(3D7), the final expressedproduct referred to as FMP-1 (SEQ ID NO:7).

[0105] The present invention also relates to a composition comprisingpeptides or polypeptides as described above, for in vitro detection ofmalaria antibodies present in a biological sample.

[0106] The present invention also relates to a composition comprising atleast one of the following MSP-1₄₂ conformational epitopes:

[0107] epitope recognized by monoclonal antibodies 12.10, 12.8, 7.5,2.2, 1E1 (Blackman et al., 1990, supra; Conway et al., 1991,Parasitology 103,1-6; McBride et al., 1982, Science 217, 254-257; Mackayet al., 1985, Embo J. 4, 3823-3829).

[0108] epitope recognized by monoclonal antibody 5.2 (Chang et al.,1988, Exp. Parasitol. 67, 1-11), and

[0109] epitope recogized by monoclonal antibody 7F1 (Lyon et al., 1987,J. Immunol. 138, 895-901).

[0110] The present invention also relates to an MSP-1₄₂ specificantibody raised upon immunizing an animal with a peptide or proteincomposition, with said antibody being specifically reactive with any ofthe polypeptides or peptides as defined above, and with said antibodybeing preferably a monoclonal antibody.

[0111] The present invention also relates to an MSP-1₄₂ specificantibody screened from a variable chain library in plasmids or phages orfrom a population of human B-cells by means of a process known in theart, with said antibody being reactive with any of the polypeptides orpeptides as defined above, and with said antibody being preferably amonoclonal antibody.

[0112] The MSP-1₄₂ specific monoclonal antibodies of the invention canbe produced by any hybridoma liable to be formed according to classicalmethods from splenic or lymph node cells of an animal, particularly froma mouse or rat, immunized against the Plasmodium polypeptides orpeptides according to the invention, as defined above on the one hand,and of cells of a myeloma cell line on the other hand, and to beselected by the ability of the hybridoma to produce the monoclonalantibodies recognizing the polypeptides which has been initially usedfor the immunization of the animals.

[0113] The antibodies involved in the invention can be labelled by anappropriate label of the enzymatic, fluorescent, or radioactive type.

[0114] The monoclonal antibodies according to this preferred embodimentof the invention may be humanized versions of mouse monoclonalantibodies made by means of recombinant DNA technology, departing fromparts of mouse and/or human genomic DNA sequences coding for H and Lchains from cDNA or genomic clones coding for H and L chains.

[0115] Alternatively the monoclonal antibodies according to thispreferred embodiment of the invention may be human monoclonalantibodies. These antibodies according to the present embodiment of theinvention can also be derived from human peripheral blood lymphocytes ofpatients infected with malaria, or vaccinated against malaria. Suchhuman monoclonal antibodies are prepared, for instance, by means ofhuman peripheral blood lymphocytes (PBL) repopulation of severe combinedimmune deficiency (SCID) mice, or by means of transgenic mice in whichhuman immunoglobulin genes have been used to replace the mouse genes.

[0116] The invention also relates to the use of the proteins or peptidesof the invention, for the selection of recombinant antibodies by theprocess of repertoire cloning.

[0117] Antibodies directed to peptides or single or specific proteinsderived from a certain strain may be used as a medicament, moreparticularly for incorporation into an immunoassay for the detection ofPlasmodium strains for detecting the presence of MSP-1₄₂ antigens, orantigens containing MSP-1₄₂ epitopes, for prognosing/monitoring ofmalaria disease, or as therapeutic agents.

[0118] Alternatively, the present invention also relates to the use ofany of the above-specified MSP-1₄₂ monoclonal antibodies for thepreparation of an immunoassay kit for detecting the presence of MSP-1₄₂antigen or antigens containing MSP-1₄₂ epitopes in a biological sample,for the preparation of a kit for prognosing/monitoring of malariadisease or for the preparation of a malaria medicament.

[0119] The present invention also relates to a method for in vitrodiagnosis or detection of malaria antigen present in a biologicalsample, comprising at least the following steps:

[0120] (i) contacting said biological sample with any of the MSP-1₄₂specific monoclonal antibodies as defined above, preferably in animmobilized form under appropriate conditions which allow the formationof an immune complex,

[0121] (ii) removing unbound components,

[0122] (iii) incubating the immune complexes formed with heterologousantibodies, which specifically bind to the antibodies present in thesample to be analyzed, with said heterologous antibodies conjugated to adetectable label under appropriate conditions,

[0123] (iv) detecting the presence of said immune complexes visually ormechanically (e.g. by means of densitometry, fluorimetry, colorimetry).

[0124] The present invention also relates to a kit for in vitrodiagnosis of a malaria antigen present in a biological sample,comprising:

[0125] at least one monoclonal antibody as defined above, with saidantibody being preferentially immobilized on a solid substrate,

[0126] a buffer or components necessary for producing the bufferenabling binding reaction between these antibodies and the malariaantigens present in the biological sample, and

[0127] a means for detecting the immune complexes formed in thepreceding binding reaction.

[0128] The kit can possibly also include an automated scanning andinterpretation device for inferring the malaria antigens present in thesample from the observed binding pattern.

[0129] Monoclonal antibodies according to the present invention aresuitable both as therapeutic and prophylactic agents for treating orpreventing malaria infection in susceptible malaria-infected subjects.Subjects include rodents such as mice or guinea pigs, monkeys, and othermammals, including humans.

[0130] In general, this will comprise administering a therapeutically orprophylactically effective amount of one or more monoclonal antibodiesof the present invention to a susceptible subject or one exhibitingmalaria infection. Any active form of the antibody can be administered,including Fab and F(ab′)₂ fragments. Antibodies of the present inventioncan be produced in any system, including insect cells, baculovirusexpression systems, chickens, rabbits, goats, cows, or plants such astomato, potato, banana or strawberry. Methods for the production ofantibodies in these systems are known to a person with ordinary skill inthe art. Preferably, the antibodies used are compatible with therecipient species such that the immune response to the MAbs does notresult in clearance of the MAbs before parasite can be controlled, andthe induced immune response to the MAbs in the subject does not induce“serum sickness” in the subject. Preferably, the MAbs administeredexhibit some secondary functions such as binding to Fc receptors of thesubject.

[0131] Treatment of individuals having malaria infection may comprisethe administration of a therapeutically effective amount of MSP-1₄₂antibodies of the present invention. The antibodies can be provided in akit as described below. The antibodies can be used or administered as amixture, for example in equal amounts, or individually, provided insequence, or administered all at once. In providing a patient withantibodies, or fragments thereof, capable of binding to MSP-1₄₂, or anantibody capable of protecting against malaria in a recipient patient,the dosage of administered agent will vary depending upon such factorsas the patient's age, weight, height, sex, general medical condition,previous medical history, etc.

[0132] In general, it is desirable to provide the recipient with adosage of antibody which is in the range of from about 1 pg/kg-100pg/kg, 100 pg/kg-500 pg/kg, 500 pg/kg-1 ng/kg, 1 ng/kg-100 ng/kg, 100ng/kg-500 ng/kg, 500 ng/kg-1 ug/kg, 1 ug/kg-100 ug/kg, 100 ug/kg-500ug/kg, 500 ug/kg-1 mg/kg, 1 mg/kg-50 mg/kg, 50 mg/kg-100 mg/kg, 100mg/kg-500 mg/kg, 500 mg/kg-1 g/kg, 1 g/kg-5 g/kg, 5 g/kg-10 g/kg (bodyweight of recipient), although a lower or higher dosage may beadministered.

[0133] In a similar approach, another prophylactic use of the monoclonalantibodies of the present invention is the active immunization of apatient using an anti-idiotypic antibody raised against one of thepresent monoclonal antibodies. Immunization with an anti-idiotype whichmimics the structure of the epitope could elicit an active anti-MSP-1₄₂response (Linthicum, D. S. and Farid, N. R., Anti-Idiotypes, Receptors,and Molecular Mimicry (1988), pp 1-5 and 285-300).

[0134] Likewise, active immunization can be induced by administering oneor more antigenic and/or immunogenic epitopes as a component of asubunit vaccine. Vaccination could be performed orally or parenterallyin amounts sufficient to enable the recipient to generate protectiveantibodies against this biologically functional region, prophylacticallyor therapeutically. The host can be actively immunized with theantigenic/immunogenic peptide in pure form, a fragment of the peptide,or a modified form of the peptide. One or more amino acids, notcorresponding to the original protein sequence can be added to the aminoor carboxyl terminus of the original peptide, or truncated form ofpeptide. Such extra amino acids are useful for coupling the peptide toanother peptide, to a large carrier protein, or to a support. Aminoacids that are useful for these purposes include: tyrosine, lysine,glutamic acid, aspartic acid, cyteine and derivatives thereof.Alternative protein modification techniques may be used e.g.,NH₂-acetylation or COOH-terminal amidation, to provide additional meansfor coupling or fusing the peptide to another protein or peptidemolecule or to a support.

[0135] The antibodies capable of protecting against malaria are intendedto be provided to recipient subjects in an amount sufficient to effect areduction in the malaria infection symptoms. An amount is said to besufficient to “effect” the reduction of infection symptoms if thedosage, route of administration, etc. of the agent are sufficient toinfluence such a response. Responses to antibody administration can bemeasured by analysis of subject's vital signs.

[0136] The present invention more particularly relates to a compositioncomprising at least one of the above-specified peptides or a recombinantMSP-1₄₂ protein composition as defined above, for use as a vaccine forimmunizing a mammal, preferably humans, against malaria, comprisingadministering a sufficient amount of the composition possiblyaccompanied by pharmaceutically acceptable adjuvant(s), to produce animmune response.

[0137] Immunogenic compositions can be prepared according to methodsknown in the art. The present compositions comprise an immunogenicamount of a recombinant MSP-1₄₂ proteins or peptides as defined above,usually combined with a pharmaceutically acceptable carrier, preferablyfurther comprising an adjuvant.

[0138] The proteins of the present invention, preferably purifiedMSP-1₄₂ derived from pETATPfMBP-1₄₂ (3D7) or FMP-1, are expected toprovide a particularly useful vaccine antigen, since the antigen is ableto induce invasion inhibitory antibodies as well as high titerantibodies that react with schizont-infected erythrocytes.

[0139] Pharmaceutically acceptable carriers include any carrier thatdoes not itself induce the production of antibodies harmful to theindividual receiving the composition. Suitable carriers are typicallylarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers; and inactive virus particles. Suchcarriers are well known to those of ordinary skill in the art.

[0140] Preferred adjuvants to enhance effectiveness of the compositioninclude adjuvants listed below with formulation for one human vaccinedose of 0.5 ml. the dosages of components described below are preferreddosages; variations in dosages that do not affect the function of theadjuvant are also encompassed by the present invention as can be readilydetermined by the person skilled in the art.

[0141] Adjuvant A, described in WO 96/33739, with the formulation 0.25mg cholesterol, 1 mg dioleoyl phosphotidylcholine, 50 ug 3D-MPL, and 50ug QS21 and consisting of small liposomes wherein the QS21 and the3D-MPL are in the membranes of the liposomes;

[0142] Adjuvant B, described in U.S. Pat. No. 6,146,632, with theformulation 10.68 mg squalene, 11.86 mg tocopherol, 4.85 mg Tween 80, 50ug 3D-MPL, and 50 ug QS21 and consisting of an oil-in water emulsioncomprising the squalene and alpha-tocopherol, the emulsion being inadmixture with the QS21 and 3-DPML;

[0143] Adjuvant C, described in WO 96/33739, with the formulation 0.25mg cholesterol, 1 mg dioleoyl phosphotidylcholine, 50 ug 3D-MPL, 50 ugQS21 and 0.5 mg AlOH₃ and consisting of small liposomes wherein thesaponin (QS21) and the LPS-derivative (3D-MPL) are in the membranes ofthe liposomes and wherein the liposomes and the antigen are absorbedonto a metallic salt particle carrier (AlOH₃);

[0144] Adjuvant D, with the formulation 0.5 mg AlOH₃, 500 ug ofunmethylated immunostimulatory oligonucleotide CpG described in WO96/02555 (CpG=5′-tcg tcg ttt tgt cgt ttt gtc gtt) (SEQ ID NO:8) whereantigen and immunostimulant (CpG) are absorbed onto a metallic saltparticle carrier (AlOH₃);

[0145] Adjuvant E, described in WO 96/33739, with the formulation 0.25mg cholesterol, 1 mg dioleoyl phosphotidylcholine, 50 ug QS21, and 0.5mg AlOH₃, consisting of small unilamellar vesicles wherein the saponin(QS21) is in the membranes of the vesicles and wherein the vesicles andthe antigen are absorbed onto a metallic salt particle AlOH₃.

[0146] All documents cited herein are hereby incoporated by referencethereto.

[0147] The immunogenic compositions typically will containpharmaceutically acceptable vehicles, such as water, saline, glycerol,ethanol, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, preservatives, and thelike, may be included in such vehicles.

[0148] Typically, the immunogenic compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid vehicles prior toinjection may also be prepared. The preparation also may be emulsifiedor encapsulated in liposomes for enhanced adjuvant effect. The MSP-1₄₂proteins of the invention may also be incorporated into ImmuneStimulating Complexes together with saponins, for example QuilA(ISCOMS).

[0149] Immunogenic compositions used as vaccines comprise a ‘sufficientamount’ or ‘an immunologically effective amount’ of the proteins of thepresent invention, as well as any other of the above mentionedcomponents, as needed. ‘Immunologically effective amount’, means thatthe administration of that amount to an individual, either in a singledose or as part of a series, is effective for treatment, as definedabove. This amount varies depending upon the health and physicalcondition of the individual to be treated, the taxonomic group ofindividual to be treated (e.g. nonhuman primate, primate, etc.), thecapacity of the individual's immune system to synthesize antibodies, thedegree of protection desired, the formulation of the vaccine, thetreating doctor's assessment of the medical situation, the strain ofmalaria infection, and other relevant factors. It is expected that theamount will fall in a relatively broad range that can be determinedthrough routine trials. Usually, the amount will vary from 0.01 to 1000ug/dose, more particularly from about 1.0 to 100 ug/dose most preferablyfrom about 10 to 50 ug/dose.

[0150] The proteins may also serve as vaccine carriers to presenthomologous (e.g. other malaria antigens, such as EBA-175 or AMA-1) orheterologous (non-malaria) antigens. In this use, the proteins of theinvention provide an immunogenic carrier capable of stimulating animmune response to other antigens. The antigen may be conjugated eitherby conventional chemical methods, or may be cloned into the geneencoding MSP-1₄₂ fused to the 5′end or the 3′ end of the MSP-1₄₂ gene.The vaccine may be administered in conjunction with otherimmunoregulatory agents.

[0151] The compounds of the present invention can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby these materials, or their functional derivatives,are combined in admixture with a phamaceutically acceptable carriervehicle. Suitable vehicles and their formulation, inclusive of otherhuman proteins, e.g., human serum albumin, are described, for example,in Remington's Pharmaceutical Sciences (16th ed., Osol, A. ed., MackEaston Pa. (1980)). In order to form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the above-described compoundstogether with a suitable amount of carrier vehicle.

[0152] Additional pharmaceutical methods may be employed to control theduration of action. Control release preparations may be achieved throughthe use of polymers to complex or absorb the compounds. The controlleddelivery may be exercised by selecting appropriate macromolecules (forexample polyesters, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine sulfate) and the concentration of macromolecules as well asthe method of incorporation in order to control release. Anotherpossible method to control the duration of action by controlled releasepreparations is to incorporate the compounds of the present inventioninto particles of a polymeric material such as polyesters, polyaminoacids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.Alternatively, instead of incorporating these agents into polymericparticles, it is possible to entrap these materials in microcapsulesprepared, for example, interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate)-microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980).

[0153] Administration of the compounds, whether antibodies or vaccines,disclosed herein may be carried out by any suitable means, includingparenteral injection (such as intraperitoneal, subcutaneous, orintramuscular injection), in ovo injection of birds, orally, or bytopical application of the antibodies (typically carried in apharmaceutical formulation) to an airway surface. Topical application ofthe antibodies to an airway surface can be carried out by intranasaladministration (e.g., by use of dropper, swab, or inhaler which depositsa pharmaceutical formulation intranasally). Topical application of theantibodies to an airway surface can also be carried out by inhalationadministration, such as by creating respirable particles of apharmaceutical formulation (including both solid particles and liquidparticles) containing the antibodies as an aerosol suspension, and thencausing the subject to inhale the respirable particles. Methods andapparatus for administering respirable particles of pharmaceuticalformulations are well known, and any conventional technique can beemployed. Oral administration may be in the form of an ingestable liquidor solid formulation.

[0154] The treatment may be given in a single dose schedule, orpreferably a multiple dose schedule in which a primary course oftreatment may be with 1-10 separate doses, followed by other doses givenat subsequent time intervals required to maintain and or reinforce theresponse, for example, at 1-4 months for a second dose, and if needed, asubsequent dose(s) after several months. Examples of suitable treatmentschedules include: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1month, (iii) 0 and 1 month, (iv) 0 and 6 months, or other schedulessufficient to elicit the desired responses expected to reduce diseasesymptoms, or reduce severity of disease.

[0155] The present invention also provides kits which are useful forcarrying out the present invention. The present kits comprise a firstcontainer means containing the above-described antibodies. The kit alsocomprises other container means containing solutions necessary orconvenient for carrying out the invention. The container means can bemade of glass, plastic or foil and can be a vial, bottle, pouch, tube,bag, etc. The kit may also contain written information, such asprocedures for carrying out the present invention or analyticalinformation, such as the amount of reagent contained in the firstcontainer means. The container means may be in another container means,e.g. a box or a bag, along with the written information.

[0156] The present invention also relates to a method for in vitrodiagnosis of malaria antibodies present in a biological sample,comprising at least the following steps

[0157] (i) contacting said biological sample with a compositioncomprising any of the MSP-1₄₂ peptides as defined above, preferably inan immobilized form under appropriate conditions which allow theformation of an immune complex, wherein said peptide or protein can be abiotinylated peptide or protein which is covalently bound to a solidsubstrate by means of streptavidin or avidin complexes,

[0158] (ii) removing unbound components,

[0159] (iii) incubating the immune complexes formed with heterologousantibodies, with said heterologous antibodies having conjugated to adetectable label under appropriate conditions,

[0160] (iv) detecting the presence of said immune complexes visually ormechanically (e.g. by means of densitometry, fluorimetry, colorimetry).

[0161] The present invention also relates to a kit for determining thepresence of malaria antibodies, in a biological sample, comprising:

[0162] at least one peptide or protein composition as defined above,possibly in combination with other polypeptides or peptides fromPlasmodium or other types of malaria parasite, with said peptides orproteins being preferentially immobilized on a solid support, morepreferably on different microwells of the same ELISA plate, and evenmore preferentially on one and the same membrane strip,

[0163] a buffer or components necessary for producing the bufferenabling binding reaction between these polypeptides or peptides and theantibodies against malaria present in the biological sample,

[0164] means for detecting the immune complexes formed in the precedingbinding reaction,

[0165] possibly also including an automated scanning and interpretationdevice for inferring the malaria parasite present in the sample from theobserved binding pattern.

[0166] The immunoassay methods according to the present inventionutilize MSP-1₄₂ domains that maintain linear (in case of peptides) andconformational epitopes (proteins) recognized by antibodies in the serafrom individuals infected with a malaria parasite. The MSP-1₄₂ antigensof the present invention may be employed in virtually any assay formatthat employs a known antigen to detect antibodies. A common feature ofall of these assays is that the antigen is contacted with the bodycomponent suspected of containing malaria antibodies under conditionsthat permit the antigen to bind to any such antibody present in thecomponent. Such conditions will typically be physiologic temperature, pHand ionic strenght using an excess of antigen. The incubation of theantigen with the specimen is followed by detection of immune complexescomprised of the antigen.

[0167] Design of the immunoassays is subject to a great deal ofvariation, and many formats are known in the art. Protocols may, forexample, use solid supports, or immunoprecipitation. Most assays involvethe use of labeled antibody or polypeptide; the labels may be, forexample, enzymatic, fluorescent, chemiluminescent, radioactive, or dyemolecules. Assays which amplify the signals from the immune complex arealso known; examples of which are assays which utilize biotin and avidinor streptavidin, and enzyme-labeled and mediated immunoassays, such asELISA assays.

[0168] The immunoassay may be, without limitation, in a heterogeneous orin a homogeneous format, and of a standard or competitive type. In aheterogeneous format, the polypeptide is typically bound to a solidmatrix or support to facilitate separation of the sample from thepolypeptide after incubation. Examples of solid supports that can beused are nitrocellulose (e.g., in membrane or microtiter well form),polyvinyl chloride (e.g., in sheets or microtiter wells), polystyrenelatex (e.g., in beads or microtiter plates, polyvinylidine fluoride(known as Immunolon.TM.), diazotized paper, nylon membranes, activatedbeads, and Protein A beads. For example, Dynatech Immunolon.TM.1 orImmunlon.TM. 2 microtiter plates or 0.25 inch polystyrene beads(Precision Plastic Ball) can be used in the heterogeneous format. Thesolid support containing the antigenic polypeptides is typically washedafter separating it from the test sample, and prior to detection ofbound antibodies. Both standard and competitive formats are know in theart.

[0169] In a homogeneous format, the test sample is incubated with thecombination of antigens in solution. For example, it may be underconditions that will precipitate any antigen-antibody complexes whichare formed. Both standard and competitive formats for these assays areknown in the art.

[0170] In a standard format, the amount of malaria antibodies in theantibody-antigen complexes is directly monitored. This may beaccomplished by determining whether labeled anti-xenogeneic (e.g.anti-human) antibodies which recognize an epitope on anti-malariaantibodies will bind due to complex formation. In a competitive format,the amount of malaria antibodies in the sample is deduced by monitoringthe competitive effect on the binding of a known amount of labeledantibody (or other competing ligand) in the complex.

[0171] Complexes formed comprising anti-malaria antibody (or in the caseof competitive assays, the amount of competing antibody) are detected byany of a number of known techniques, depending on the format. Forexample, unlabeled malaria antibodies in the complex may be detectedusing a conjugate of anti-xenogeneic Ig complexed with a label (e.g. anenzyme label).

[0172] In an immunoprecipitation or agglutination assay format thereaction between the malaria antigens and the antibody forms a networkthat precipitates from the solution or suspension and forms a visiblelayer or film of precipitate. If no anti-malaria antibody is present inthe test specimen, no visible precipitate is formed.

[0173] There currently exist three specific types of particleagglutination (PA) assays. These assays are used for the detection ofantibodies to various antigens when coated to a support. One type ofthis assay is the hemagglutination assay using red blood cells (RBCs)that are sensitized by passively adsorbing antigen (or antibody) to theRBC. The addition of specific antigen antibodies present in the bodycomponent, if any, causes the RBCs coated with the purified antigen toagglutinate.

[0174] To eliminate potential non-specific reactions in thehemagglutination assay, two artificial carriers may be used instead ofRBC in the PA. The most common of these are latex particles. However,gelatin particles may also be used. The assays utilizing either of thesecarriers are based on passive agglutination of the particles coated withpurified antigens.

[0175] The MSP-1₄₂ proteins, peptides, or antigens of the presentinvention will typically be packaged in the form of a kit for use inthese immunoassays. The kit will normally contain in separate containersthe MSP-1₄₂ antigen, control antibody formulations (positive and/ornegative), labeled antibody when the assay format requires the same andsignal generating reagents (e.g. enzyme substrate) if the label does notgenerate a signal directly. The MSP-1₄₂ antigen may be already bound toa solid matrix or separate with reagents for binding it to the matrix.Instructions (e.g. written, tape, CD-ROM, etc.) for carrying out theassay usually will be included in the kit.

[0176] Immunoassays that utilize the MSP-1₄₂ antigen are useful inscreening blood for the preparation of a supply from which potentiallyinfective malaria parasite is lacking. The method for the preparation ofthe blood supply comprises the following steps. Reacting a bodycomponent, preferably blood or a blood component, from the individualdonating blood with MSP-1₄₂ proteins of the present invention to allowan immunological reaction between malaria antibodies, if any, and theMSP-1₄₂ antigen. Detecting whether anti-malaria antibody—MSP-1₄₂ antigencomplexes are formed as a result of the reacting. Blood contributed tothe blood supply is from donors that do not exhibit antibodies to thenative MSP-1 antigens.

[0177] The present invention further contemplates the use of MSP-1₄₂proteins, or parts thereof as defined above, for in vitro monitoringmalaria infection or prognosing the response to treatment (for instancewith chloroquine, mefloquine, Malarome) of patients suffering frommalaria infection comprising:

[0178] incubating a biological sample from a patient with malariainfection with an MSP-1₄₂ protein or a suitable part thereof underconditions allowing the formation of an immunological complex,

[0179] removing unbound components,

[0180] calculating the anti-MSP-1₄₂ titers present in said sample (forexample at the start of and/or during the course of therapy),

[0181] monitoring the natural course of malaria infection, or prognosingthe response to treatment of said patient on the basis of the amountanti-MSP-1₄₂ titers found in said sample at the start of treatmentand/or during the course of treatment.

[0182] Patients who show a decrease of 2, 3, 4, 5, 7, 10, 15, orpreferably more than 20 times of the initial anti-MSP-1₄₂ titers couldbe concluded to be long-term, sustained responders to malaria therapy.

[0183] It is to be understood that smaller fragments of theabove-mentioned peptides also fall within the scope of the presentinvention. Said smaller fragments can be easily prepared by chemicalsynthesis and can be tested for their ability to be used in an assay asdetailed above.

[0184] The present invention also relates to a kit for monitoringmalaria infection or prognosing the response to treatment (for instanceto medication) of patients suffering from malaria infection comprising:

[0185] at least one MSP-1₄₂ peptide as defined above,

[0186] a buffer or components necessary for producing the bufferenabling the binding reaction between these proteins or peptides and theanti-MSP-1₄₂ antibodies present in a biological sample,

[0187] means for detecting the immune complexes formed in the precedingbinding reaction,

[0188] possibly also an automated scanning and interpretation device forinferring a decrease of anti-MSP-1₄₂ titers during the progression oftreatment.

[0189] The present invention also relates to a serotyping assay fordetecting one or more serological types or alleles of malaria parasitepresent in a biological sample, more particularly for detectingantibodies of the different types or alleles of malaria parasites to bedetected combined in one assay format, comprising at least the followingsteps:

[0190] (i) contacting the biological sample to be analyzed for thepresence of malaria antibodies of one or more serological types, with atleast one of the MSP-1₄₂ compositions as defined above, preferentiallyin an immobilized form under appropriate conditions which allow theformation of an immune complex,

[0191] (ii) removing unbound components,

[0192] (iii) incubating the immune complexes formed with heterolagousantibodies, with said heterologous antibodies being conjugated to adetectable label under appropriate conditions,

[0193] (iv) detecting the presence of said immune complexes visually ormechanically (e.g. by means of densitometry, fluorimetry, calorimetry)and inferring the presence of one or more malaria serological typespresent from the observed binding pattern.

[0194] It is to be understood that the compositions of proteins orpeptides used in this method are recombinantly expressed type-specificor allele-specific proteins or type-specific peptides.

[0195] The present invention further relates to a kit for serotyping oneor more serological types or alleles of malaria parasite present in abiological sample, more particularly for detecting the antibodies tothese serological types of malaria parasites comprising:

[0196] at least one MSP-1₄₂ protein or MSP-1₄₂ peptide, as definedabove,

[0197] a buffer or components necessary for producing the bufferenabling the binding reaction between these proteins or peptides and theanti-MSP-1 antibodies present in a biological sample,

[0198] means for detecting the immune complexes formed in the precedingbinding reaction,

[0199] possibly also an automated scanning and interpretation device fordetecting the presence of one or more serological types present from theobserved binding pattern.

[0200] The present invention also relates to the use of a peptide orprotein composition as defined above, for immobilization on a solidsupport and incorporation into a reversed phase hybridization assay,preferably for immobilization as parallel lines onto a solid supportsuch as a membrane strip, for determining the presence or the genotypeof malaria parasite according to a method as defined above. Combinationwith other type-specific or allele-specific antigens from other malariaparasites also lies within the scope of the present invention.

[0201] The contents of all cited references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application arehereby expressly incorporated by reference.

[0202] Other features of the invention will become apparent in thecourse of the following descriptions of exemplary embodiments which aregiven for illustration of the invention and are not intended to belimiting thereof.

[0203] The following MATERIALS AND METHODS were used in the examplesthat follow.

[0204] Construction of Expression Cassette pET(AT)PfMSP-1₄₂ (3D7)

[0205] Molecular cloning and bacterial transformations were performed asdescribed (Sambrook et al., Molecular cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, 1989). The final expression construct,pETATPfMSP-142 (3D7) Tolle et al., 1995, Exp. Parasitol. 81, 47-54, wasthe product of a series of subclonings, each successive constructionreducing the amount of expressed non-MSP-1 sequence. The MSP-1₄₂fragment was prepared by PCR of genomic DNA using the forward primerGGGGATCCATTGAGGGTCGTGGTACCATGGC AATATCTGTCACAATGG (SEQ ID NO:9) and thereverse primer GTCGACTTAGGAACTGCAGAAAATACCGG (SEQ ID NO:10). The productwas cloned into the expression vector pMAL-p (New England Biolabs) viathe 5′ BamHI and the 3′ SalI site, sequenced (SEQ ID NO:4), and thensubcloned into pET32a (Novagen, Madison, Wis.), creating pET-Trx42,which contained the MSP-1₄₂ gene fragment fused in-frame to the 3′ endof the E. coli thioredoxin gene, trx. pET-Trx42 was digested with NdeIand religated to remove trx, creating pET(50)MSP-1₄₂. pET42A was createdby digesting both the pET(50)MSP-1₄₂ vector and the DNA fragmentGGGCATATGGCACACCATCATCATCATCATCCCGGGGGATCCGAC (SEQ ID NO: 11) with NdeIand BamHI and then ligating the two. The DNA fragment encoded sixconsecutive histidine residues and a short flexible linker sequence. Toavoid using ampicillin selection, tet was subcloned from pBR322 bydigesting with EcoRI and PflMI, then blunt-ending and ligating it intothe Bst1107 I site in pET42A creating pET-IEGR-MSP-1₄₂ (AT). The finalplasmid pET(AT)PfMSP-1₄₂(3D7) was created by removing the residualFactor Xa cleavage site. This was accomplished by digestingpET-IEGR-MSP-1₄₂ (AT) and the DNA fragmentGGGCATATGGCACACCATCATCATCATCATCCCGGGGGAT CCGGTTCTGGTACCGAC (SEQ IDNO:12), with NdeI and KpnI and then ligating. 3D7 MSP-1₄₂ was expressedfrom this final construct with 17 non-MSP-1₄₂ amino acids fused to theN-terminus.

[0206] Expression of MSP-1₄₂.

[0207] The expression host, BL21 DE3 (F-ompT hsdSB(rB-mB-) gal dcm(DE3)) was transformed with pETATPfMSP-1₄₂(3D7). Fresh stationary phasecultures of transformed bacteria were used to inoculate 1L shake flasksof Super Broth containing 15 ug/ml tetracycline, which were grown to 0.4OD 600 at 37° C. cooled to temperatures ranging from 25-35° C. andinduced with 0.1 mM IPTG. Cell pastes were collected in lysis buffer (10mM NaPO₄, pH 6.2, 50 mM NaCl, 10 mM imidazole, 2 mM MgCl₂, 50 U/mlbenzonase) at a paste to buffer ratio of 1:3 w/v. Cells were lysed bymicrofluidization (Microfluidics) in one pass, NaCl was added to a finalconcentration of 500 mM. The temperature of the sample was maintainedbelow 10° C. at all times. Tween-80, 1.0% w/v (final concentration), wasadded and the lysate was centrifuged at 27,666×g for 1 hr at 4° C.Pellets and supernates were evaluated by immunoblotting.

[0208] Bulk Fermentation and Expression of MSP-1₄₂ (3D7).

[0209] A 300 L fermentor of Super Broth supplemented with 15 ug/mltetracycline was inoculated with three L of fresh stationary phaseculture in accordance with Batch Production Record (BPR)-305-00.Fermentation continued until an OD 600=4.0-6.0 was reached. Thefermentor was cooled to 25° C. prior to induction with 0.1 mM IPTG.Three hours following induction, cells were harvested by centrifugationat 15,000 rpm at 3 L/min. The cell paste was stored at −80° C. in theWRAIR Department of Biologics Research, Pilot Bioproduction Facility.

[0210] GMP Purification of E. coli expressed MSP-1₄₂ (3D7), BPR-335-O₂.Cell paste was lysed as described above. Tween-80 was added to 1% w/v(final), and the lysate was centrifuged at 27,666×g for 1 hr at 4° C.The clarified lysate was collected and placed on ice. All further stepswere carried out at 4° C.

[0211] Ni⁺² NTA Superflow (Qiagen, Germany): A column with a 6:1 w/vcell paste to resin ratio, was equilibrated with 10 mM NaPO₄, pH 6.2,500 mM NaCl, 10 mM imidazole (Ni-buffer) supplemented with 0.5% Tween 80(w/v). The clarified lysate was applied at a flow rate of 30 ml/min andthe column washed with 1.1 volumes of Ni-buffer containing 0.5%Tween-80. The column was then washed with 30 volumes of Ni-buffercontaining 0.5% Tween 80 (w/v); 20 volumes of 10 mM NaPO₄, pH 6.2, 75 mMNaCl, 10 mM imidazole; and 15 volumes of 10 mM NaPO₄, pH 8.0, 75 mMNaCl, 20 mM imidazole. MSP-1₄₂ was eluted with 10 volumes 10 mM NaPO₄,pH 8.0, 75 mM NaCl, 160 mM imidazole, and was diluted with an equalvolume of 10 mM NaPO₄, 75 mM NaCl, pH 8.0, and 0.4% Tween 80.

[0212] Q ion exchange chromatography: A Toyopearl SuperQ 650 M(TosoHaas) column (cell paste: resin ratio=3:1 w/v), was equilibratedwith 10 mM NaPO₄, pH 8.0, 75 mM NaCl, 80 mM imidazole, 0.2% Tween 80(Q-buffer). The diluted sample was applied at a flow rate of 30 ml/minand washed with one volume of Q-buffer, which was combined with the flowthrough to pool the MSP-1₄₂, giving a final volume of this pool equal to50 Q column volumes. This sample was diluted with an equal volume of 10mM NaPO₄, pH 6.0, 0.4% Tween 80 (v/v), and the pH of the sample wasadjusted to pH 6.0 with 6N HCl.

[0213] CM ion exchange chromatography: A CM 650 M (TosoHaas) column(cell paste:resin ratio=2:1) was equilibrated with 10 mM NaPO₄, pH 6.0,35 mM NaCl, 0.2% Tween 80 (CM-equilibration buffer) and the sample wasapplied at a flow rate of 30 ml/min. The column was washed with 6volumes of CM-equilibration buffer, followed by 10 volumes of 10 mMNaPO₄, pH 7.0, 100 mM NaCl, 0.02% Tween 80 (v/v). The MSP-1₄₂ was elutedwith 10 mM NaPO₄, pH 7.2, 250 mM NaCl in three column volumes.

[0214] SDS-PAGE and Immunoblottincr. Protein samples were separatedunder reducing (10% 2-mercaptoethanol) or nonreducing conditions bySDS-PAGE with Tris-Glycine buffering (Invitrogen). Protein was detectedby Coomassie Blue R250 staining. Immunoblotting was perfomed withnitrocellulose membranes (Invitrogen) blocked using 5% nonfat dry milkand 0.1% Tween 20 in PBS, pH 7.4. Blots were probed with polyclonalrabbit anti-MSP-1₄₂ antibodies or mAbs diluted into phosphate bufferedsaline, pH 7.4 containing 0.1% Tween 20. This buffer was also used forwashing. The second antibodies were alkaline phosphatase-conjugatedanti-rabbit IgG or anti-mouse IgG (H+L) (Promega, Madison, Wis.) andreactions were detected with nitro-blue tetrazolium and5-bromo-4-chloro-3-indolyl phosphate (Sigma Chemicals, St. Louis, Mo.)in 100 mM NaCl, 5 mM MgCl₂, 100 mM Tris-HCl, pH 9.5. mAbs used forevaluation of structure included 2.2 (Mackay et al., 1985, Embo J. 4,3823-3829), 12.8 (Conway et al, 1991, Parasitology 103, 1-6), 7.5(McBride et al., 1982, Science 217, 254-257), 12.10 (Blackman et al.,1990, supra), 5.2 (Chang et al., 1988, Exp. Parasitol. 67, 1-11), and7F1 (Lyon et al., 1987, J. Immunol. 138, 895-901).

[0215] Antigen Stability Studies

[0216] Stability studies were conducted for 18 months on MSP-1₄₂ storedat 4° C., −20° C., and −80° C. and for lyophilized product stored at−20° C. Stability was evaluated by Commassie Blue staining andimmunoblotting of SDS PAGE gels run under non-reduced and reducedconditions.

[0217] Vaccine Preparation

[0218] The CM eluate was concentrated two-fold to 0.5 mg/ml, the bufferwas exchanged with 10 volumes of phosphate buffered saline bydiafiltration and the protein was sterilized by filtration through aMillipak 60 0.22-um filtration unit. Final bulk antigen (FMP1) wasstored at −800C. To prepare the antigen for use with adjuvant ADJUVANT B(GlaxoSmithKline Biologicals, Rixensart, Belgium), 118.5 ml of sterilepurified FMP1 was mixed with 236 ml of 50 mM Na Phosphate, 101 ml of15.75% lactose, and 0.5 ml Tween-80, producing formulated antigen at aconcentration of 118.5 mg/ml. Formulated FMP1 was sterile filtered withMillipak 40 0.22-um filtration unit and added to 3 ml vials forlyophilization. The vials were sealed with Lyo stoppers and metal crimps(BPR-334-01, Lot 0678)

[0219] Vaccine Potency

[0220] Female Balb/C mice were immunized subcutaneously with 100 ul ofvaccine. Potency studies were performed with 1.0, 0.3 and 0.1 ug ofMSP-1₄₂ in Adjuvant B (GlaxoSmithKline Biologicals, Rixensart, Belgium).The 10 ug dose was prepared with 10 ug of antigen and 100 ul of AdjuvantB and was used to make the 1.0 and 0.1 ug doses by diluting into 0.9%saline. Mice were primed and then bled and boosted four-weeks later, andbled again 2 weeks following boosting. Sera were analyzed by ELISA andresults are reported in ELISA units, or the serum dilution that gives anabsorbance of 1 OD 405 (Stoute et al., 1997, N. Engl. J. Med. 336,86-91).

[0221] Seroconversion occurred if the following condition was met:(ELISAUnits-3SD)post-vaccination−(ELISA Units+3SD)pre-vaccination>0

[0222] Safety and Immunogenicity

[0223] Rhesus monkeys were vaccinated intramuscularly with 50 ug ofantigen formulated with ADJUVANT B or alum. Monkeys were boosted one,three, five, and seven months after priming and sera were collectedprior to and two weeks following each immunization.

[0224] Serology

[0225] Sera were analyzed by IFA against methanol fixed 3D7 strain P.falciparum schizont-infected erythrocytes (Lyon et al., 1987, supra) andby kinetic ELISA. For this, the MSP-1₄₂ capture antigen was diluted inPBS at pH 7.4 and coated at 0.4 pmoles/well overnight at 4° C., andwells were blocked with CaseinBlock (Pierce). Sera were diluted 1:50,000(ADJUVANT B) or 1:8,000 (Alum) in CaseinBlock and reacted for 1 h atroom temperature, followed by reaction with alkalinephosphatase-conjugated rabbit anti-human IgG (H&L) (Promega) diluted1:250 in CaseinBlock also for 1 hr at room temperature. Detection ofp-nitrophenyl phosphate substrate conversion to product was measured at5 min intervals for 30 min. The slope of the line was calculated bylinear regression and R2 was at least 0.99 for each analysis.

[0226] Inhibition of Parasite Invasion

[0227] Rabbits were vaccinated four times subcutaneously with FMP1 inFreund's adjuvants and sera were collected three weeks after the finalimmunization. The IgG fractions of pre-immune and post-immune sera wereprepared with Protein-G chromatography and quantified by using theBradford Protein Determination assay (Pierce). For the invasioninhibition assay (Chulay et al., 1981, Am. J. Trop. Med. Hyg. 30,12-19), the IgG fractions were dialyzed against RPMI 1640 adjusted to pH7.4 with NaOH and added to settled 100 ul cultures of synchronous P.falciparum (3D7 strain) schizont-infected erythrocytes (3-5 nuclei) at2% hematocrit and 0.25% parasitemia. In some experiments, native FMP1 orreduced and alkykated FMP1, dialyzed as above, were added as specificitycontrols to reverse the activity of inhibitory antibodies.

EXAMPLE 1

[0228] The gene fragment containing P. falciparum 3D7 strain MSP-1₄₂ wascloned into pET32a (FIG. 1A) with several modifications. These includedadding the gene for tetracycline, removing the trx gene and otherelements, and adding a hexa-histidine affinity tag, which contained 17non-MSP-1₄₂ amino acids fused to the N-terminus of MSP-1₄₂ (FIG. 1B).Our primary objective of expressing MSP-1₄₂ as a soluble protein in E.coli cytoplasm was achieved by systematically varying IPTG concentrationand induction temperature. IPTG at 0.1 mM induced maximal proteinsynthesis and induction at 25° C. was required to express solubleprotein (data not shown). The optimal biomass density for induction in10L fermentors was shown to be 4-6 OD600. These conditions were used toprepare GMP cell paste in a 300 L fermentor, and this paste was used todevelop the purification process. The MSP-1₄₂ was purified by threechromatographic steps. After centrifugation, cleared lysates wereapplied to a Ni⁺² NTA Superflow resin for affinity purification (FIG.2A, lane 1). This step removed most of the endotoxin, which depended onextensive washing of bound protein at low pH (pH 6.2) and high sodiumchloride (500 mM). After this step, the protein was greater than 50%pure by densitometry (arrows show MSP-1₄₂ related bands). MSP-1₄₂ waspurified to greater than 95% purity by chromatography on a Q-anionexchanger followed by a CM-cation exchanger (FIG. 2A, lanes 2 and 3respectively). The Final Bulk Antigen (FMP1) was further characterizedby SDS-PAGE under non-reducing and reducing conditions (FIG. 2B, lanes 1and 2, respectively) and by immunoblotting with rabbit anti-E. coliantibody (FIG. 2C, lane 3) and MSP-1 specific mAbs. The MSP-1₃₃ specificmAb 7F1 reacted with two proteins migrating at 36 kD and 38 kD (FIG. 2C,lane 1), but the MSP-1₁₉ specific mAb 12.10 (FIG. 2C, lane 2) did not,nor did any of the other MSP-1₁₉ specific mAbs. Full-length MSP-1₄₂ (seeFIG. 2) and all of the higher molecular weight aggregates were reactiveagainst all the MSP-1 specific mAbs used. Long term stability studiesshowed that the FMP1 was stable when stored for 18 months at −80° C. butnot when stored at 4° C. (degradation) or −20° C. (aggregation) (FIG. 3,lanes 1-3, respectively), compare with FIG. 2B (lane 1).

EXAMPLE 2

[0229] Vaccine potency studies were conducted in Balb/C mice immunizedwith FMP1 formulated for use with adjuvant ADJUVANT B. At the 0.3 ug and1.0 ug doses, respectively, all mice seroconverted following oneimmunization. At the 0.1 ug dose 50% of the mice seroconverted followingthe first immunization; all seroconverted after the second (data notshown). Safety and immunogenicity of the product was assessed in Rhesusmonkeys immunized up to five times with 50 ug doses of FMP1/ADJUVANT B(n=8) or FMP1/alum (n=6). No adverse local responses were observed andall biochemical and hematological laboratory tests were normal for bothgroups (not shown). FMP1/ADJUVANT B induced malaria parasite reactiveIFA titers that increased to 1:28,000 by two weeks following the secondimmunization and maintained this level through the third immunization(FIG. 4, right ordinate). The fourth immunization induced a briefincrease in titer, which returned to the 1:28,000 base within six weeks.The fifth immunization induced a response that was similar to thefourth. FMP1/alum induced IFA titer that paralleled those induced byFMP1/ADJUVANT B but were six-fold lower. FMP1/ADJUVANT B inducedMSP-1₄₂-specific ELISA-reactive antibodies that increased to 2100 OD/minby two weeks following the second immunization and gradually increase toa maximum of 2800 OD/min after the fourth immunization (FIG. 4, leftordinate). The geometric mean antibody level after five immunizationswas lower than after four, but this difference was not significant. Byfour weeks after each immunization antibody levels fell about 30% butstabilized after the third immunization. FMP1/alum inducedELISA-reactive antibodies levels roughly paralleled those induced byFMP1/ADJUVANT B but were about ten-fold lower. FMP1 in Freund'sadjuvants induced invasion inhibitory IgG antibodies. The inhibition wastitratible (FIG. 5) and was completely reversed by adding competingsoluble FMP1 at a final concentration of 17 ug/ml and partially reversedby adding reduced and alkylated FMPl(approximately 50% reversal) at thesame concentration (data not shown).

EXAMPLE 3

[0230] FMP1 combined with the adjuvants ADJUVANT A, ADJUVANT B ADJUVANTC, ADJUVANT E, Adjuvant D, and Alum were assessed for safety andimmunogenicity in Rhesus monkeys at the human dose of 50 ug/injection.

[0231] Studies were conducted in adult Macaca mulatta housed at theArmed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok,Thailand. Monkeys were screened to exclude animals in poor health orwith previous exposure to malaria, and were monitored for at least 6weeks prior to the start of the study. Animals were randomly assigned totreatment groups, and vaccines were administered in a blindedstandardized fashion while animals were under ketamine anesthesia.

[0232] Animals received intramuscular injections of FMP1 (50 ug)combined with adjuvant at 0, 4, 12 weeks. The attending veterinarian whoconducted clinical and laboratory evaluations assessed vaccine safety.Blood was obtained for CBC and serum chemistries prior to administrationand at 24, 48, and 72 hours immediately after each immunization. Localreactogenicity was monitored at the time of immunization, day 1, day 2,day 3, and day 14 post-immunization.

[0233] Safety

[0234] The following safety data apply to FMP1 combined with ADJUVANT B.The same data are available for the other adjuvants as well.

[0235] Clinical evaluations before and after each immunization revealedminimal local and no systemic toxicities. Reactogenicity of FMP1 inADJUVANT B was assessed in each Rhesus monkey using the followingcriteria: 1) skin warmth or calor; 2) skin erythema or rubor; 3) skinswelling or edema; 4) muscle induration; 5) muscle necrosis. Theintensity of the reactions were scored as follows: 0=none; 1=slight ormild reaction; 2=moderate reaction; 3=marked or severe reaction. Themean prevalence (Table 2A) and mean intensity (Table 2B) of localreactogenicity is given in the tables below. Erythema, skin swelling,and muscle induration were limited to the site of inoculation andresolved in all monkeys by 14 days post immunization. There were nocases of muscle necrosis. The intensity of muscle induration diminisheddaily from post immunization day 1 through day 3, and multipleimmunizations did not increase the risk of adverse reactions. Behavior,activity, and food consumption remained normal. TABLE 2A Mean Prevalenceof Reactogenicity - mean % positive (n = 8 Rhesus) Immuni- Post- SkinSkin Skin zation immuni- warmth/ erythema/ swelling/ Muscle MuscleNumber zation day calor rubor edema induration necrosis 1 10 Aug. 1999 —0% 0% 0% 0% 0% 11 Aug. 1999 1 50%  12.5%   37.5%   87.5%   0% 12 Aug.1999 2 0% 0% 12.5%   75%  0% 13 Aug. 1999 3 0% 0% 0% 12.5%   0% 24 Aug.1999 14 0% 0% 0% 0% 0% 2  7 Sep. 1999 — 0% 0 of 8 0 of 8 0 of 8 0%  8Sep. 1999 1 12.5%   87.5%   100%  100%  0%  9 Sep. 1999 2 0% 50%  100% 100%  0% 10 Sep. 1999 3 0% 12.5%   87.5%   100%  0% 21 Sep. 1999 14 0%0% 0% 0% 0% 3  9 Nov. 1999 — 0 of 8 12.5%   2 of 8 0 of 8 0% 10 Nov.1999 1 37.5%   25%  75%  100%  0% 11 Nov. 1999 2 12.5%   0% 37.5%  100%  0% 12 Nov. 1999 3 0% 0% 0% 75%  0% 23 Nov. 1999 14 0% 0% 0% 0% 0%

[0236] TABLE 2B Immuni- Post- Skin Skin Skin zation immuni- warmth/erythema/ swelling/ Muscle Muscle Number zation day calor rubor edemainduration necrosis 1 10 Aug. 1999 — 0 0 0 0 0 11 Aug. 1999 1 1 1 1.31.3 0 12 Aug. 1999 2 0 0 1 1 0 13 Aug. 1999 3 0 0 0 1 0 24 Aug. 1999 140 0 0 0 0 2  7 Sep. 1999 — 0 0 0 0 0  8 Sep. 1999 1 2 1.6 3 2.9 0  9Sep. 1999 2 0 1.8 3 2.4 0 10 Sep. 1999 3 0 1 2.6 2.3 0 21 Sep. 1999 14 00 0 0 0 3  9 Nov. 1999 — 0 0 0 0 0 10 Nov. 1999 1 1 1.5 1.3 2.1 0 11Nov. 1999 2 1 0 1 1.6 0 12 Nov. 1999 3 0 0 0 1.5 0 23 Nov. 1999 14 0 0 00 0

[0237] Mean laboratory values for the entire group (Tables 3 and datanot shown) indicated no significant abnormalities or trends inhematologic or biochemical laboratory tests. Quantitative plateletcounts varied before and after immunization. This variation occurredboth within individual Rhesus monkeys and between Rhesus. All bloodprior to December 1999 was collected in heparin anti-coagulant, and wesuspected that the platelet count variation was attributed to the use ofthe heparin anticoagulant. All Rhesus in the study had blood samplescollected in mid December 1999 using both heparin and EDTAanticoagulants and quantitative platelet counts determined at 2locations in Bangkok Thailand. AFRIMS veterinary pathologists read allblood smears from August, September, and November and the plateletcounts were read as adequate, increased, or decreased. It was decidedthat all further hematologic (CBC) laboratory assays would be collectedin EDTA. The January 2000 samples reflect this change in bloodcollection procedure with normal platelet counts observed in all Rhesusimmunized with FMP1 and ADJUVANT B days 1, 2, and 3 following a 4^(th)immunization. In addition, all blood smears at each time point reviewedafter immunizations 1, 2, and 3 had adequate platelet counts (data notshown).

[0238] Immunogenicity

[0239] Humoral Immunity: FMP1 combined with ADJUVANT A or ADJUVANT Binduced similar levels of ELISA (Table 4A) and IFA (Table 4B) reactiveantibody and were significantly more potent than the other adjuvantcombinations. FMP1 combined with Alum was the least immunogenic by ELISAafter two immunizations, but was not different from ADJUVANT C, ADJUVANTE and Adjuvant D by ELISA after the third immunization. FMP1 combinedwith Alum or with ADJUVANT C induced similar levels of IFA reactiveantibody and induced significantly more IFA reactive antibody than FMP1combined with ADJUVANT E or Adjuvant D.

[0240] Cellular Immunity: FMP1 combined with ADJUVANT A or ADJUVANT Binduced different cytokine response profiles (Table 5). Both vaccinesinduced comparable levels of IFN-γ response after the 3^(rd) dose,however, the IL-5 response was greatly suppressed in animals receivingFMP1 combined with ADJUVANT A. Duration of T cell response data indicatethat IFN-γ response generated by FMP1 combined with both ADJUVANT A andADJUVANT B persists at least 24 weeks after vaccination. The differenceon Th1/Th2 polarization of FMP1 formulated with ADJUVANT B and ADJUVANTA was not anticipated. TABLE 3 Mean Biochemical Laboratory Test ValuesImmunization #1 Immunization #2 Immunization #3 August SeptemberNovember 5 11 12 13 7 8 9 10 9 10 11 12 BUN (mg/ml) MEAN 19.7 17.3 18.119.0 20.0 16.9 20.6 17.9 17.9 18.5 16.3 16.4 s.d. 3.5 3.4 2.4 3.7 2.92.6 2.2 2.9 2.9 1.1 2.4 1.9 CREATININE (mg/ml) MEAN 0.76 0.81 0.80 0.810.80 0.83 0.75 0.81 0.88 0.90 0.83 0.86 s.d. 0.07 0.04 0.11 0.06 0.090.12 0.08 0.11 0.18 0.09 0.10 0.12 AST (U/l) MEAN 23.9 80.8 67.9 61.927.4 47.0 50.1 47.6 27.8 68.8 57.3 46.0 s.d. 3.7 38.9 22.5 24.4 4.1 13.620.3 17.1 4.5 23.3 18.5 14.9 ALT (U/l) MEAN 28.0 50.9 50.1 56.3 32.837.3 39.5 43.9 38.4 41.5 47.0 51.5 s.d. 9.1 16.0 15.2 17.1 13.3 13.612.7 12.9 10.6 13.7 12.8 13.5 CPK (U/l) MEAN 301 1192 879 923 256 791671 470 307 2443 915 520 s.d. 239 550 684 657 208 356 298 237 368 1039721 221

[0241] TABLE 4A Rhesus Humoral Responses to FMP1 formulated with variousadjuvants Midpoint ELISA Titer Week 0* Week 2 Week 4* Week 6 Week 13*Week 15 ADJUVANT A GMT 13 1402 1824 52161 9291 74348 95% CI 17 1413 117115783 4088 20937 ADJUVANT B GMT 7 2439 3929 87096 25574 98458 95% CI 2657 2164 24748 6797 42826 ADJUVANT C GMT 27 325 715 31654 2576 25120 95%CI 101 597 714 15885 633 9183 ADJUVANT E GMT 6 80 186 25360 2698 3594595% CI 3 155 234 9517 915 10550 ADJUVANT D GMT 5 245 392 16899 179923647 95% CI 24 315 259 16810 2113 12386 Alum GMT 8 64 98 5501 751 1096995% CI 21 129 118 7168 865 11011

[0242] TABLE 4B Endpoint IFA Titer Week 0* Week 2 Week 4* Week 6 Week13* Week 15 ADJUVANT A GMT 0 4000 1308 26251 11888 43069 95% CI 0 20797492 5410 6372 11853 ADJUVANT B GMT 0 3668 4362 26909 19027 26909 95% CI0 1283 1793 5132 6584 5132 ADJUVANT C GMT 0 24 917 9514 2594 10375 95%CI 488 257 849 6458 0 6187 ADJUVANT E GMT 14 12 15 144 31 257 95% CI 3 46 98 29 131 ADJUVANT D GMT 50 54 74 675 169 844 95% CI 13 19 21 319 66238 Alum GMT 0 316 794 2828 1414 4490 95% CI 0 470 0 4158 839 3719

[0243] TABLE 5 Polarization by ADJUVANT A of Immune Rosponses to FMP1Adjuvant IFN-γ/IL-5 ADJUVANT A 4.2 ADJUVANT B 0.75 ADJUVANT C 2.3ADJUVANT E 0.77 Adjuvant D 5.0 Alum 0.53

EXAMPLE 4

[0244] A Phase I dose escalation clinical trial of the recombinantPlasmodium falciparum malaria vaccine candidate FMP1/ADJUVANT B wasrecently completed at the Walter Reed Army Institute of Research (WRAIR)in Silver Spring, Md. in 15 adult human volunteers to assess safety,reactogenicity, and immunogenicity. This vaccine was created byresearchers at the WRAIR and manufactured at the Pilot BioproductionFacility at WRAIR. In the initial Phase I clinical trial conductedduring 4th quarter 2000—1st quarter 2001, three groups of 5 volunteerswere immunized with ⅕th dose, ½ dose, or full dose of vaccine at 0, 1,and 3 months. Tables 1 and 2 below summarize the demographics of thestudy population.

[0245] Safety and Adverse Events:

[0246] Local and general systemic adverse events were assessed at 6 timepoints following each immunization, and blood tests to evaluatehematologic, renal, and hepatic abnormalities were performed before andafter each immunization. The vaccine proved safe in all 15 volunteerswith NO serious adverse events or clinical laboratory abnormalitiesnoted. There were no drop-outs from the vaccine trial. All adverseevents were graded according to severity. There were no Serious AdverseEvents (SAE's) requiring hospitalization nor were there any grade 3adverse events as defined below. Grade “3”=Adverse experience whichprevents normal everyday activities and necessitates a correctivetherapy. The specific occurances by subject and by dose of all adverseevents are summarized in tables 3-7 below. In summary, the most frequentadverse events was minimal pain at the site of vaccine injection whichdisappeared by 24 hours post-inoculation.

[0247] Immunogenicity:

[0248] ELISA:

[0249] The vaccine was extremely potent in inducing high-titer antibodyresponses in all volunteers as assessed by ELISA (enzyme-linkedimmunoabsorbent assay). The table below summarizes the mean, standarddeviation, and geometric mean antibody titers for each of the threevaccine groups. TABLE 6 ANTIBODY TITERS to MSP-1₄₂ BY ELISA VALUESINDICATE DILUTION OF SERA WHICH GIVES OD = 1 Day 0 Day 14 Day 28 Day 42Day 84 Day 98 ⅕ Dose Average 12 312 462 18066 7371 32648 Std Dev 8 191236 8406 6304 24046 Geo Mean 10 272 412 16440 5749 26626 ½ DOSE Average28 1285 2530 44172 ND 57771 Std Dev 6 1882 2631 21176 ND 24192 Geo Mean28 636 1762 40744 ND 53569 Full Dose Average 32 688 990 32461 1591450053 Std Dev 32 414 306 19307 5650 29991 Geo Mean 22 586 951 2844814938 42799

[0250] IFA: The vaccine was also immunogenic as assessed by indirectimmunofluorescence titers to malaria parasites. TABLE 7IMMUNOFLUORESCENCE to 3D7 Parasites Values indicate serum dilution whichgives 1 + IFA intensity to methanol-fixed malaria parasites DAY OF STUDYDay 0 Day 14 Day 28 Day 42 Day 84 Day 98 ⅕ Dose-GROUP 1 Geomean 200 200200 1393 2111 3676 95% CI 1152 2533 5155 SD 0 0 0 1315 2890 5881 ½Dose-GROUP 2 Geomean 200 200 200 2425 ND 5572 95% CI 768 ND 1254 SD 0 00 876 ND 1431 Full Dose-GROUP 3 Geomean 200 200 200 3200 3676 8445 95%CI 1536 1882 3967 SD 0 0 0 1753 2147 4525

[0251] In addition, cell-mediated immunologic responses were noted inthe majority of vaccinated subjects. Peripheral blood mononuclear cellswere stimulated with MSP-1 antigen or P. falciparum parasitizederythrocytes and proliferation was measured by uptake of ³H-thymidine.FIG. 6 shows the results of PBMC proliferation in each of the subjectsafter each dose of vaccine.

[0252] Summary

[0253] In this initial clinical trial involving a small number ofvolunteers, FMP1/ADJUVANT B has been shown to be a safe and highlyimmunogenic vaccine that elicits both parasite-reactive antibodies andcellular responses. This study develops a foundation to further test itssafety profile and evaluate its efficacy to reduce morbidity andmortality in target populations directly affect by P. falciparummalaria.

[0254] Safety Data TABLE 8 Number of subjects TOTAL Percent Group 1Group 2 Group 3 Number of subjects 15 100% 5 5 5 planned Subjects orvaccine 0 0 0 0 0 number not allocated Number of subjects 15 (100%) 5 55 enrolled (Total cohort)

[0255] TABLE 9 Demographics: Study population Mean Age Min age Max. AgeS.D. Sex N (years) (years) (years) (years) Female 5 32.2 27 51 10.8 Male10 34.4 22 52 11.1 Total 15 33.7 22 52 10.7

[0256] TABLE 10 Incidence and nature of symptoms reported per dose andper subject after vaccination General symptoms Local symptoms Dose GroupN n % n % By dose Dose 1 15 2 13 12 80 Dose 2 15 2 13 11 73 Dose 3 15 17 10 67 Overall 45 5 11 33 73 By volunteer Group 1 5 1 20 4 80 Group 2 51 20 4 80 Group 3 5 2 40 5 100

[0257] TABLE 11 Incidence of solicited local symptoms including symptomsgraded at maximum intensity Group 1 Group 2 Group 3 Solicited local (N =15) (N = 15) (N = 15) Symptom Intensity n % n % n % Pain Total 8 53.3 1173.3 14 93.3 grade “3” 0 0 0 0 0 0 Redness Total 1 6.7 4 26.7 2 13.3 >50mm/>24 h 0 0 0 0 0 0 Swelling Total 0 0 0 0 0 0 >50 mm/>24 h 0 0 0 0 0 0

[0258] TABLE 12 Subjects reporting solicited local symptoms includingsymptoms graded at maximum intensity Group 1 Group 2 Group 3 Solicitedlocal (N = 5) (N = 5) (N = 5) symptom Intensity n % n % n % Pain Total 480 4 80 5 100 grade “3” 0 0 0 0 0 0 Redness Total 1 20 3 60 2 40 >50mm/>24 h 0 0 0 0 0 0 Swelling Total 0 0 0 0 0 0 >50 mm/>24 h 0 0 0 0 0 0

[0259] TABLE 13 Incidence of solicited general symptoms includingsymptoms graded at maximum intensity and those probably or suspected ofbeing related to vaccination Group 1 Group 2 Group 2 N = 15 N = 15 N =15 Symptoms N % N % N % Arthralgia Total 0 0 0 0 1 6.7 PB/SU 0 0 0 0 0 0PB/SU & 0 0 0 0 0 0 Grade “3” Fever Total 0 0 0 0 1 6.7 PB/SU 0 0 0 0 00 PB/SU & 0 0 0 0 0 0 Grade “3” Headache Total 0 0 0 0 0 0 PB/SU 0 0 0 00 0 PB/SU & 0 0 0 0 0 0 Grade “3” Malaise Total 1 6.7 1 6.7 1 6.7 PB/SU1 6.7 1 6.7 0 0 PB/SU & 0 0 0 0 0 0 Grade “3” Myalgia Total 1 6.7 0 0 16.7 PB/SU 1 6.7 0 0 0 0 PB/SU & 0 0 0 0 0 0 Grade “3” Rash Total 0 0 0 00 0 PB/SU 0 0 0 0 0 0 PB/SU & 0 0 0 0 0 0 Grade “3” Dizziness Total 0 00 0 1 6.7 PB/SU 0 0 0 0 0 0 PB/SU & 0 0 0 0 0 0 Grade “3” Nausea Total 00 0 0 1 6.7 PB/SU 0 0 0 0 0 0 PB/SU & 0 0 0 0 0 0 Grade “3”

[0260] TABLE 14 Subjects reporting solicited general symptoms includingsymptoms graded at maximum intensity and those probably or suspected ofbeing related to vaccination Group 1 Group 2 (Full dose) (⅕ dose) (½dose) Group 2 N = 5 N = 5 N = 5 Symptoms N % N % N % Arthralgia Total 00 0 0 1 20 PB/SU 0 0 0 0 0 0 PB/SU & Grade “3” 0 0 0 0 0 0 Fever Total 00 0 0 1 20 PB/SU 0 0 0 0 0 0 PB/SU & Grade “3” 0 0 0 0 0 0 HeadacheTotal 0 0 0 0 0 0 PB/SU 0 0 0 0 0 0 PB/SU & Grade “3” 0 0 0 0 0 0Fatigue Total 1 20 1 20 1 20 PB/SU 1 20 1 20 0 0 PB/SU & Grade “3” 0 0 00 0 0 Myalgia Total 1 20 0 0 1 20 PB/SU 20 0 0 0 0 0 PB/SU & Grade “3” 00 0 0 0 0 Rash Total 0 0 0 0 0 0 PB/SU 0 0 0 0 0 0 PB/SU & Grade “3” 0 00 0 0 0 Dizziness Total 0 0 0 0 1 20 PB/SU 0 0 0 0 0 0 PB/SU & Grade “3”0 0 0 0 0 0 Nausea Total 0 0 0 0 1 20 PB/SU 0 0 0 0 0 0 PB/SU & Grade“3” 0 0 0 0 0 0

[0261] EXAMPLE 5

[0262] Three rabbits per immunization group were vaccinated 4 times at3-week intervals with 50 μg FMP1 (3D7) in Montamide adjuvant,subcutaneously, or with FMP1 in ADJUVANT B, intramuscularly. Twonegative control rabbits per group were immunized with each adjuvantalone. A final control rabbit was immunized with reduced and alkylatedMSP1₄₂ (3D7) in Montamide. Each rabbit was bled from the ear vein 2weeks following each immunization. Following the fourth immunization therabbits were ex-sanguinated from the heart and the sera from theserabbits was analyzed by MSP1 (3D7)-specific antigen ELISA's.

[0263] The sera were analyzed by MSP1 (3D7)-specific ELISA and bykinetic ELISA. The MSP1-specific capture antigen was diluted in PBS atpH 7.4 and coated at 0.4 pmoles/well overnight at 4° C. and the wellswere blocked with CaseinBlock (Pierce). Sera were first diluted by 1:25and then followed by two-fold serial dilutions down the plate up to1.6×10⁶ fold. Sera were reacted for 1 hour at room temperature, followedby reaction with alkaline phosphatase-conjugated goat anti-rabbit IgG(H&L)(Promega) diluted 1:5,000 in CaseinBlock for 1 hour at roomtemperature. Detection of p-nitrophenyl phosphate substrate conversionto product was measured at 60 minutes. The data are reported as theaverage of triplicate values plotted from the titration curve measuredat OD₄₀₅.

[0264] Immunization with FMP1 in Montamide and FMP1 in ADJUVANT Binduces high MSP1₄₂ specific antibody titers following the secondimmunization. The geometric mean of the post fourth immunization MSP1₄₂specific antibody titers induced by immunization with FMP1/ADJUVANT Band FMP1/Montamide were 1:363,000 and 1:182,000, respectively. Neitheradjuvant/antigen combination substantially boosted the MSP1₄₂ specificantibody titers after a third and fourth immunization.

[0265] Discussion

[0266] Here we describe the development, fermentation, expression,purification and characterization of a safe and immunogenic recombinantMSP-1₄₂ that is suitable for production of antibodies and for use indiagnostic assays and as a potential vaccine.

[0267] MSP-1₄₂ which was derived from recombinant E. coli was highlypurified and met all FDA standards necessary for testing safety andimmunogenicity in humans. Endotoxin levels were significantly below theFDA acceptable levels (FDA; 350 EU/dose/70 kg human, Our Process; 9.14EU/dose/70 kg human). Residual levels of all chemicals used in thepurification process were quantified and determined to be within levelsset as production specifications. Although the MSP-1₄₂ comprised greaterthan 95% of the protein in the final product, it was not homogeneous dueto proteolysis at the C-terminal end of the protein. This proteolysisappeared to occur during expression in the E. coli host because longterm studies showed that the antigen was stable when stored at −80° C.for 18 months, revealing little change in the Coomassie Blue stainingpattern or mAb reaction patterns in immunoblots (FIG. 3).

[0268] Previous studies have shown that the induction of antibodyresponses to epitopes on MSP-1₁₉ correlate with clinical immunity tomalaria (Egan et al., 1996, J. Infect. Dis. 173, 765-769), suggestingthat induction of such responses depends on correctly forming thedisulfide-dependent conformational epitopes present within the MSP-1₁₉portion of MSP-1₄₂. This conclusion is further supported by theobservation that the only MSP-1₁₉ specific mAbs capable of inhibitionmerozoite invasion react with conformational epitopes (Burghaus et al.,1994, Mol. Biochem. Parasitol. 64, 165-169). Recombinant MSP-1₄₂ hascorrect structure because it reacted with all of the MSP-1₁₉ specificmAbs we used; these were raised against malaria parasites and includedfunctional mAbs classified as growth or invasion inhibitory (mAb 12.10,12.8) (Blackman et al., 1990, supra) and blocking inhibitory mAbs (mAb7.5, 2.2, 1E1) (Guevara et al., 1997, J. Exp. Med. 186, 1689-1699).

[0269] These data support the continued use of bacterial systems forexpressing soluble malaria antigens that contain conformational epitopesas potential vaccine candidates.

[0270] In addition, FMP1/ADJUVANT B was highly immunogenic in the Rhesusmonkeys as well as Balb/C mice. After three immunizations the geometricmean IFA antibody titers induced in Rhesus monkeys exceeded 1:36,000 andthe geometric mean ELISA titer exceed 26,000 OD/min (FIG. 4); thesetiters did not change with further immunization. FMP1/ADJUVANT B inducedapproximately six times more MSP-1 specific antibodies than FMP1/Alum(FIG. 4). MSP-1 specific antibodies induced by FMP1 were predominantlyagainst MSP-1₄₂ and MSP-1₁₉ compared to MSP-1₃₃ (not shown). Thisresult, when taken in combination with FMP1's ability to induce hightiter antibodies that react with schizont-infected erythrocytes and it'sability to induce invasion inhibitory antibodies (FIG. 5 and Table 1)further indicates that it has correct structure. Currently, the onlymalaria vaccine that has reproducibly protected human volunteers is theRTS,S/ADJUVANT B vaccine (Kester et al., 2001, J.

[0271] Infect. Dis. 183, 640-647). When combined with ADJUVANT B, FMP1was highly immunogenic and caused no adverse biochemical or localreactions in mice.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 12 <210> SEQ ID NO 1<211> LENGTH: 546 <212> TYPE: PRT <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: E. coli expressed P. falciparumMSP142 (3D7) Protein Sequence in pET-Trx42 <400> SEQUENCE: 1 Met Ser AspLys Ile Ile His Leu Thr Asp 1 5 10 Asp Ser Phe Asp Thr Asp Val Leu LysAla 15 20 Asp Gly Ala Ile Leu Val Asp Phe Trp Ala 25 30 Glu Trp Cys GlyPro Cys Lys Met Ile Ala 35 40 Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr 4550 Gln Gly Lys Leu Thr Val Ala Lys Leu Asn 55 60 Ile Asp Gln Asn Pro GlyThr Ala Pro Lys 65 70 Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 75 80 LeuPhe Lys Asn Gly Glu Val Ala Ala Thr 85 90 Lys Val Gly Ala Leu Ser lysGly Gln Leu 95 100 Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly 105 110 SerGly Ser Gly His Met His His His His 115 120 His His Ser Ser Gly Leu ValPro Arg Gly 125 130 Ser Gly Met Lys Glu Thr Ala Ala Ala Lys 135 140 PheGlu Arg Gln His Met Asp Ser Pro Asp 145 150 Leu Gly Thr Asp Asp Asp AspLys Ala Met 155 160 Ala Asp Ile Gly Ser Ile Glu Gly Arg Gly 165 170 ThrMet Ala Ile Ser Val Thr Met Asp Asn 175 180 Ile Leu Ser Gly Phe Glu AsnGlu Tyr Asp 185 190 Val Ile Tyr Leu Lys Pro Leu Ala Gly Val 195 200 TyrArg Ser Leu Lys Lys Gln Ile Glu Lys 205 210 Asn Ile Phe Thr Phe Asn LeuAsn Leu Asn 215 220 Asp Ile Leu Asn Ser Arg Leu Lys Lys Arg 225 230 LysTyr Phe Leu Asp Val Leu Glu Ser Asp 235 240 Leu Met Gln Phe Lys His IleSer Ser Asn 245 250 Glu Tyr Ile Ile Glu Asp Ser Phe Lys Leu 255 260 LeuAsn Ser Glu Gln Lys Asn Thr Leu Leu 265 270 Lys Ser Tyr Lys Tyr Ile LysGlu Ser Val 275 280 Glu Asn Asp Ile Lys Phe Ala Gln Glu Gly 285 290 IleSer Tyr Tyr Glu Lys Val Leu Ala Lys 295 300 Tyr Lys Asp Asp Leu Glu SerIle Lys Lys 305 310 Val Ile Lys Glu Glu Lys Glu Lys Phe Pro 315 320 SerSer Pro Pro Thr Thr Pro Pro Ser Pro 325 330 Ala Lys Thr Asp Glu Gln LysLys Glu Ser 335 340 Lys Phe Leu Pro Phe Leu Thr Asn Ile Glu 345 350 ThrLeu Tyr Asn Asn Leu Val Asn Lys Ile 355 360 Asp Asp Tyr Leu Ile Asn LeuLys Ala Lys 365 370 Ile Asn Asp Cys Asn Val Glu Lys Asp Glu 375 380 AlaHis Val Lys Ile Thr Lys Leu Ser Asp 385 390 Leu Lys Ala Ile Asp Asp LysIle Asp Leu 395 400 Phe Lys Asn Pro Tyr Asp Phe Glu Ala Ile 405 410 LysLys Leu Ile Asn Asp Asp Thr Lys Lys 415 420 Asp Met Leu Gly Lys Leu LeuSer Thr Gly 425 430 Leu Val Gln Asn Phe Pro Asn Thr Ile Ile 435 440 SerLys Leu Ile Glu Gly Lys Phe Gln Asp 445 450 Met Leu Asn Ile Ser Gln HisGln Cys Val 455 460 Lys Lys Gln Cys Pro Glu Asn Ser Gly Cys 465 470 PheArg His Leu Asp Glu Arg Glu Glu Cys 475 480 Lys Cys Leu Leu Asn Tyr LysGln Glu Gly 485 490 Asp Lys Cys Val Glu Asn Pro Asn Pro Thr 495 500 CysAsn Glu Asn Asn Gly Gly Cys Asp Ala 505 510 Asp Ala Thr Cys Thr Glu GluAsp Ser Gly 515 520 Ser Ser Arg Lys Lys Ile Thr Cys Glu Cys 525 530 ThrLys Pro Asp Ser Tyr Pro Leu Phe Asp 535 540 Gly Ile Phe Cys Ser Ser 545<210> SEQ ID NO 2 <211> LENGTH: 431 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: E. coliexpressed P. falciparum MSP142 (3D7) Protein Sequence in pET(50)MSP1-42<400> SEQUENCE: 2 Met His His His His His His Ser Ser Gly 1 5 10 Leu ValPro Arg Gly Ser Gly Met Lys Glu 15 20 Thr Ala Ala Ala Lys Phe Glu ArgGln His 25 30 Met Asp Ser Pro Asp Leu Gly Thr Asp Asp 35 40 Asp Asp LysAla Met Ala Asp Ile Gly Ser 45 50 Ile Glu Gly Arg Gly Thr Met Ala IleSer 55 60 Val Thr Met Asp Asn Ile Leu Ser Gly Phe 65 70 Glu Asn Glu TyrAsp Val Ile Tyr Leu Lys 75 80 Pro Leu Ala Gly Val Tyr Arg Ser Leu Lys 8590 Lys Gln Ile Glu Lys Asn Ile Phe Thr Phe 95 100 Asn Leu Asn Leu AsnAsp Ile Leu Asn Ser 105 110 Arg Leu Lys Lys Arg Lys Tyr Phe Leu Asp 115120 Val Leu Glu Ser Asp Leu Met Gln Phe Lys 125 130 His Ile Ser Ser AsnGlu Tyr Ile Ile Glu 135 140 Asp Ser Phe Lys Leu Leu Asn Ser Glu Gln 145150 Lys Asn Thr Leu Leu Lys Ser Tyr Lys Tyr 155 160 Ile Lys Glu Ser ValGlu Asn Asp Ile Lys 165 170 Phe Ala Gln Glu Gly Ile Ser Tyr Tyr Glu 175180 Lys Val Leu Ala Lys Tyr Lys Asp Asp Leu 185 190 Glu Ser Ile Lys LysVal Ile Lys Glu Glu 195 200 Lys Glu Lys Phe Pro Ser Ser Pro Pro Thr 205210 Thr Pro Pro Ser Pro Ala Lys Thr Asp Glu 215 220 Gln Lys Lys Glu SerLys Phe Leu Pro Phe 225 230 Leu Thr Asn Ile Glu Thr Leu Tyr Asn Asn 235240 Leu Val Asn Lys Ile Asp Asp Tyr Leu Ile 245 250 Asn Leu Lys Ala LysIle Asn Asp Cys Asn 255 260 Val Glu Lys Asp Glu Ala His Val Lys Ile 265270 Thr Lys Leu Ser Asp Leu Lys Ala Ile Asp 275 280 Asp Lys Ile Asp LeuPhe Lys Asn Pro Tyr 285 290 Asp Phe Glu Ala Ile Lys Lys Leu Ile Asn 295300 Asp Asp Thr Lys Lys Asp Met Leu Gly Lys 305 310 Leu Leu Ser Thr GlyLeu Val Gln Asn Phe 315 320 Pro Asn Thr Ile Ile Ser Lys Leu Ile Glu 325330 Gly Lys Phe Gln Asp Met Leu Asn Ile Ser 335 340 Gln His Gln Cys ValLys Lys Gln Cys Pro 345 350 Glu Asn Ser Gly Cys Phe Arg His Leu Asp 355360 Glu Arg Glu Glu Cys Lys Cys Leu Leu Asn 365 370 Tyr Lys Gln Glu GlyAsp Lys Cys Val Glu 375 380 Asn Pro Asn Pro Thr Cys Asn Glu Asn Asn 385390 Gly Gly Cys Asp Ala Asp Ala Thr Cys Thr 395 400 Glu Glu Asp Ser GlySer Ser Arg Lys Lys 405 410 Ile Thr Cys Glu Cys Thr Lys Pro Asp Ser 415420 Tyr Pro Leu Phe Asp Gly Ile Phe Cys Ser 425 430 Ser <210> SEQ ID NO3 <211> LENGTH: 393 <212> TYPE: PRT <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: E. coli expressed P. falciparumMSP142 (3D7) Protein Sequence in pET42A <400> SEQUENCE: 3 Met Ala HisHis His His His His Pro Gly 1 5 10 Gly Ser Ile Glu Gly Arg Gly Thr MetAla 15 20 Ile Ser Val Thr Met Asp Asn Ile Leu Ser 25 30 Gly Phe Glu AsnGlu Tyr Asp Val Ile Tyr 35 40 Leu Lys Pro Leu Ala Gly Val Tyr Arg Ser 4550 Leu Lys Lys Gln Ile Glu Lys Asn Ile Phe 55 60 Thr Phe Asn Leu Asn LeuAsn Asp Ile Leu 65 70 Asn Ser Arg Leu Lys Lys Arg Lys Tyr Phe 75 80 LeuAsp Val Leu Glu Ser Asp Leu Met Gln 85 90 Phe Lys His Ile Ser Ser AsnGlu Tyr Ile 95 100 Ile Glu Asp Ser Phe Lys Leu Leu Asn Ser 105 110 GluGln Lys Asn Thr Leu Leu Lys Ser Tyr 115 120 Lys Tyr Ile Lys Glu Ser ValGlu Asn Asp 125 130 Ile Lys Phe Ala Gln Glu Gly Ile Ser Tyr 135 140 TyrGlu Lys Val Leu Ala Lys Tyr Lys Asp 145 150 Asp Leu Glu Ser Ile Lys LysVal Ile Lys 155 160 Glu Glu Lys Glu Lys Phe Pro Ser Ser Pro 165 170 ProThr Thr Pro Pro Ser Pro Ala Lys Thr 175 180 Asp Glu Gln Lys Lys Glu SerLys Phe Leu 185 190 Pro Phe Leu Thr Asn Ile Glu Thr Leu Tyr 195 200 AsnAsn Leu Val Asn Lys Ile Asp Asp Tyr 205 210 Leu Ile Asn Leu Lys Ala LysIle Asn Asp 215 220 Cys Asn Val Glu Lys Asp Glu Ala His Val 225 230 LysIle Thr Lys Leu Ser Asp Leu Lys Ala 235 240 Ile Asp Asp Lys Ile Asp LeuPhe Lys Asn 245 250 Pro Tyr Asp Phe Glu Ala Ile Lys Lys Leu 255 260 IleAsn Asp Asp Thr Lys Lys Asp Met Leu 265 270 Gly Lys Leu Leu Ser Thr GlyLeu Val Gln 275 280 Asn Phe Pro Asn Thr Ile Ile Ser Lys Leu 285 290 IleGlu Gly Lys Phe Gln Asp Met Leu Asn 295 300 Ile Ser Gln His Gln Cys ValLys Lys Gln 305 310 Cys Pro Glu Asn Ser Gly Cys Phe Arg His 315 320 LeuAsp Glu Arg Glu Glu Cys Lys Cys Leu 325 330 Leu Asn Tyr Lys Gln Glu GlyAsp Lys Cys 335 340 Val Glu Asn Pro Asn Pro Thr Cys Asn Glu 345 350 AsnAsn Gly Gly Cys Asp Ala Asp Ala Thr 355 360 Cys Thr Glu Glu Asp Ser GlySer Ser Arg 365 370 Lys Lys Ile Thr Cys Glu Cys Thr Lys Pro 375 380 AspSer Tyr Pro Leu Phe Asp Gly Ile Phe 385 390 Cys Ser Ser <210> SEQ ID NO4 <211> LENGTH: 1140 <212> TYPE: DNA <213> ORGANISM: Plasmodiumfalciparum 3D7 MSP142 <400> SEQUENCE: 4 ggtaccatgg caatatctgt cacaatggataatatcctct 40 caggatttga aaatgaatat gatgttatat atttaaaacc 80 tttagctggagtatatagaa gcttaaaaaa acaaattgaa 120 aaaaacattt ttacatttaa tttaaatttgaacgatatct 160 taaattcacg tcttaagaaa cgaaaatatt tcttagatgt 200attagaatct gatttaatgc aatttaaaca tatatcctca 240 aatgaataca ttattgaagattcatttaaa ttattgaatt 280 cagaacaaaa aaacacactt ttaaaaagtt acaaatatat320 aaaagaatca gtagaaaatg atattaaatt tgcacaggaa 360 ggtataagttattatgaaaa ggttttagcg aaatataagg 400 atgatttaga atcaattaaa aaagttatcaaagaagaaaa 440 ggagaagttc ccatcatcac caccaacaac acctccgtca 480ccagcaaaaa cagacgaaca aaagaaggaa agtaagttcc 520 ttccattttt aacaaacattgagaccttat acaataactt 560 agttaataaa attgacgatt acttaattaa cttaaaggca600 aagattaacg attgtaatgt tgaaaaagat gaagcacatg 640 ttaaaataactaaacttagt gatttaaaag caattgatga 680 caaaatagat ctttttaaaa accctaccgacttcgaagca 720 attaaaaaat tgataaatga tgatacgaaa aaagatatgc 760ttggcaaatt acttagtaca ggattagttc aaatttttcc 800 taatacaata atatcaaaattaattgaagg aaaattccaa 840 gatatgttaa acatttcaca acaccaatgc gtaaaaaaac880 aatgtccaga aaattctgga tgtttcagac atttagatga 920 aagagaagaatgtaaatgtt tattaaatta caaacaagaa 960 ggtgataaat gtgttgaaaa tccaaatcctacttgtaacg 1000 aaaataatgg tggatgtgat gcagatgcca catgtaccga 1040agaagattca ggtagcagca gaaagaaaat cacatgtgaa 1080 tgtactaaac ctgattcttatccacttttc gatggtattt 1120 tctgcagttc ctaagtcgac 1140 <210> SEQ ID NO 5<211> LENGTH: 383 <212> TYPE: PRT <213> ORGANISM: Plasmodium falciparum3D7 MSP142 <400> SEQUENCE: 5 Gly Ser Ile Glu Gly Arg Gly Thr Met Ala 1 510 Ile Ser Val Thr Met Asp Asn Ile Leu Ser 15 20 Gly Phe Glu Asn Glu TyrAsp Val Ile Tyr 25 30 Leu Lys Pro Leu Ala Gly Val Tyr Arg Ser 35 40 LeuLys Lys Gln Ile Glu Lys Asn Ile Phe 45 50 Thr Phe Asn Leu Asn Leu AsnAsp Ile Leu 55 60 Asn Ser Arg Leu Lys Lys Arg Lys Tyr Phe 65 70 Leu AspVal Leu Glu Ser Asp Leu Met Gln 75 80 Phe Lys His Ile Ser Ser Asn GluTyr Ile 85 90 Ile Glu Asp Ser Phe Lys Leu Leu Asn Ser 95 100 Glu Gln LysAsn Thr Leu Leu Lys Ser Tyr 105 110 Lys Tyr Ile Lys Glu Ser Val Glu AsnAsp 115 120 Ile Lys Phe Ala Gln Glu Gly Ile Ser Tyr 125 130 Tyr Glu LysVal Leu Ala Lys Tyr Lys Asp 135 140 Asp Leu Glu Ser Ile Lys Lys Val IleLys 145 150 Glu Glu Lys Glu Lys Phe Pro Ser Ser Pro 155 160 Pro Thr ThrPro Pro Ser Pro Ala Lys Thr 165 170 Asp Glu Gln Lys Lys Glu Ser Lys PheLeu 175 180 Pro Phe Leu Thr Asn Ile Glu Thr Leu Tyr 185 190 Asn Asn LeuVal Asn Lys Ile Asp Asp Tyr 195 200 Leu Ile Asn Leu Lys Ala Lys Ile AsnAsp 205 210 Cys Asn Val Glu Lys Asp Glu Ala His Val 215 220 Lys Ile ThrLys Leu Ser Asp Leu Lys Ala 225 230 Ile Asp Asp Lys Ile Asp Leu Phe LysAsn 235 240 Pro Thr Asp Phe Glu Ala Ile Lys Lys Leu 245 250 Ile Asn AspAsp Thr Lys Lys Asp Met Leu 255 260 Gly Lys Leu Leu Ser Thr Gly Leu ValGln 265 270 Ile Phe Pro Asn Thr Ile Ile Ser Lys Leu 275 280 Ile Glu GlyLys Phe Gln Asp Met Leu Asn 285 290 Ile Ser Gln His Gln Cys Val Lys LysGln 295 300 Cys Pro Glu Asn Ser Gly Cys Phe Arg His 305 310 Leu Asp GluArg Glu Glu Cys Lys Cys Leu 315 320 Leu Asn Tyr Lys Gln Glu Gly Asp LysCys 325 330 Val Glu Asn Pro Asn Pro Thr Cys Asn Glu 335 340 Asn Asn GlyGly Cys Asp Ala Asp Ala Thr 345 350 Cys Thr Glu Glu Asp Ser Gly Ser SerArg 355 360 Lys Lys Ile Thr Cys Glu Cys Thr Lys Pro 365 370 Asp Ser TyrPro Leu Phe Asp Gly Ile Phe 375 380 Cys Ser Ser <210> SEQ ID NO 6 <211>LENGTH: 1176 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: E. coli expressed P. falciparumMSP-142 (3D7) <400> SEQUENCE: 6 atggcacacc atcatcatca tcatcccgggggatccggtt 40 ctggtaccat ggcaatatct gtcacaatgg ataatatcct 80 ctcaggatttgaaaatgaat atgatgttat atatttaaaa 120 cctttagctg gagtatatag aagcttaaaaaaacaaattg 160 aaaaaaacat ttttacattt aatttaaatt tgaacgatat 200cttaaattca cgtcttaaga aacgaaaata tttcttagat 240 gtattagaat ctgatttaatgcaatttaaa catatatcct 280 caaatgaata cattattgaa gattcattta aattattgaa320 ttcagaacaa aaaaacacac ttttaaaaag ttacaaatat 360 ataaaagaatcagtagaaaa tgatattaaa tttgcacagg 400 aaggtataag ttattatgaa aaggttttagcgaaatataa 440 ggatgattta gaatcaatta aaaaagttat caaagaagaa 480aaggagaagt tcccatcatc accaccaaca acacctccgt 520 caccagcaaa aacagacgaacaaaagaagg aaagtaagtt 560 ccttccattt ttaacaaaca ttgagacctt atacaataac600 ttagttaata aaattgacga ttacttaatt aacttaaagg 640 caaagattaacgattgtaat gttgaaaaag atgaagcaca 680 tgttaaaata actaaactta gtgatttaaaagcaattgat 720 gacaaaatag atctttttaa aaacccttac gacttcgaag 760caattaaaaa attgataaat gatgatacga aaaaagatat 800 gcttggcaaa ttacttagtacaggattagt tcaaaatttt 840 cctaatacaa taatatcaaa attaattgaa ggaaaattcc880 aagatatgtt aaacatttca caacaccaat gcgtaaaaaa 920 acaatgtccagaaaattctg gatgtttcag acatttagat 960 gaaagagaag aatgtaaatg tttattaaattacaaacaag 1000 aaggtgataa atgtgttgaa aatccaaatc ctacttgtaa 1040cgaaaataat ggtggatgtg atgcagatgc cacatgtacc 1080 gaagaagatt caggtagcagcagaaagaaa atcacatgtg 1120 aatgtactaa acctgattct tatccacttt tcgatggtat1160 tttctgcagt tcctaa 1176 <210> SEQ ID NO 7 <211> LENGTH: 391 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: E. coli expressed P. falciparum MSP-142 (3D7) <400>SEQUENCE: 7 Met Ala His His His His His His Pro Gly 1 5 10 Gly Ser GlySer Gly Thr Met Ala Ile Ser 15 20 Val Thr Met Asp Asn Ile Leu Ser GlyPhe 25 30 Glu Asn Glu Tyr Asp Val Ile Tyr Leu Lys 35 40 Pro Leu Ala GlyVal Tyr Arg Ser Leu Lys 45 50 Lys Gln Ile Glu Lys Asn Ile Phe Thr Phe 5560 Asn Leu Asn Leu Asn Asp Ile Leu Asn Ser 65 70 Arg Leu Lys Lys Arg LysTyr Phe Leu Asp 75 80 Val Leu Glu Ser Asp Leu Met Gln Phe Lys 85 90 HisIle Ser Ser Asn Glu Tyr Ile Ile Glu 95 100 Asp Ser Phe Lys Leu Leu AsnSer Glu Gln 105 110 Lys Asn Thr Leu Leu Lys Ser Tyr Lys Tyr 115 120 IleLys Glu Ser Val Glu Asn Asp Ile Lys 125 130 Phe Ala Gln Glu Gly Ile SerTyr Tyr Glu 135 140 Lys Val Leu Ala Lys Tyr Lys Asp Asp Leu 145 150 GluSer Ile Lys Lys Val Ile Lys Glu Glu 155 160 Lys Glu Lys Phe Pro Ser SerPro Pro Thr 165 170 Thr Pro Pro Ser Pro Ala Lys Thr Asp Glu 175 180 GlnLys Lys Glu Ser Lys Phe Leu Pro Phe 185 190 Leu Thr Asn Ile Glu Thr LeuTyr Asn Asn 195 200 Leu Val Asn Lys Ile Asp Asp Tyr Leu Ile 205 210 AsnLeu Lys Ala Lys Ile Asn Asp Cys Asn 215 220 Val Glu Lys Asp Glu Ala HisVal Lys Ile 225 230 Thr Lys Leu Ser Asp Leu Lys Ala Ile Asp 235 240 AspLys Ile Asp Leu Phe Lys Asn Pro Tyr 245 250 Asp Phe Glu Ala Ile Lys LysLeu Ile Asn 255 260 Asp Asp Thr Lys Lys Asp Met Leu Gly Lys 265 270 LeuLeu Ser Thr Gly Leu Val Gln Asn Phe 275 280 Pro Asn Thr Ile Ile Ser LysLeu Ile Glu 285 290 Gly Lys Phe Gln Asp Met Leu Asn Ile Ser 295 300 GlnHis Gln Cys Val Lys Lys Gln Cys Pro 305 310 Glu Asn Ser Gly Cys Phe ArgHis Leu Asp 315 320 Glu Arg Glu Glu Cys Lys Cys Leu Leu Asn 325 330 TyrLys Gln Glu Gly Asp Lys Cys Val Glu 335 340 Asn Pro Asn Pro Thr Cys AsnGlu Asn Asn 345 350 Gly Gly Cys Asp Ala Asp Ala Thr Cys Thr 355 360 GluGlu Asp Ser Gly Ser Ser Arg Lys Lys 365 370 Ile Thr Cys Glu Cys Thr LysPro Asp Ser 375 380 Tyr Pro Leu Phe Asp Gly Ile Phe Cys Ser 385 390 Ser<210> SEQ ID NO 8 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: CpGoligonucleotide <400> SEQUENCE: 8 tcgtcgtttt gtcgttttgt cgtt 24 <210>SEQ ID NO 9 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer <400>SEQUENCE: 9 ggggatccat tgagggtcgt ggtaccatgg caatatctgt 40 cacaatgg 48<210> SEQ ID NO 10 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer<400> SEQUENCE: 10 gtcgacttag gaactgcaga aaataccgg 29 <210> SEQ ID NO 11<211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 11gggcatatgg cacaccatca tcatcatcat cccgggggat 40 ccgac 45 <210> SEQ ID NO12 <211> LENGTH: 57 <212> TYPE: DNA <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 12gggcatatgg cacaccatca tcatcatcat cccgggggat 40 ccggttctgg taccgac 57

What is claimed is:
 1. A vaccine against malaria comprising P.falciparum MSP-1₄₂ and an adjuvant selected from the group consisting ofA, B, C, D, and E.
 2. A vaccine according to claim 1 wherein said P.falciparum is 3D7.
 3. A method for inducing in a subject an immuneresponse against malaria infection comprising administering to saidsubject a composition comprising an immunologically effective amount ofP. falciparum MSP-1₄₂ in an acceptable diluent and an adjuvant chosenfrom the group consisting of A, B, C, D, and E.
 4. The method of claim 3wherein said P. falciparum is 3D7.
 5. A method for inducing a protectiveimmune response to malaria in a mammal, comprising administering acomposition comprising a P. falciparum MSP-1₄₂ in an amount effective toinduce an immune response in said mammal and an adjuvant selected fromthe group consisting of A, B, C, D, and E.
 6. The method of claim 5wherein said P. falciparum is 3D7.
 7. The method of claim 5, wherein thecomposition is administered to the individual in an amount of 50 ug perdose.
 8. The method of claim 5, wherein the composition is administeredparenterally.
 9. The method of claim 5, wherein the composition isadministered intranasally.
 10. The method of claim 5, wherein saidadministration is a multiple administration.
 11. The method according toclaim 10 wherein said multiple administration is at 0 and 6 months.